Sulfonylethyl phosphorodiamidates

ABSTRACT

Sulfonylethyl and thioethyl phosphorodiamidates, their preparation and intermediates in their preparation, formulations containing them, and their pharmaceutical use. The compounds are useful for treating cancer, alone and in combination with other anticancer therapies.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. 119(e) of U.S.Provisional Applications Nos. 60/573,532, filed 21 May 2004, and No.60/588,436, filed 16 Jul. 2004, both of which are incorporated into thisapplication by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to sulfonylethyl and thioethylphosphorodiamidates, formulations containing them, their pharmaceuticaluse, and their preparation and intermediates in their preparation.

2. Description of the Related Art

U.S. Pat. No. 5,556,942 [and PCT Publication No. WO 95/09865] disclosescompounds of the formula

and their amides, esters, and salts, where:

-   -   L is an electron withdrawing leaving group;    -   S^(x) is (═O)—, —S(═O)₂—, —S(═NH)—, —S(═O)(═NH)—, —S⁺(C₁-C₆        alkyl)—, —Se(═O)—, —Se(═O)₂, —Se(═NH)—, or —Se(═O)(═NH)—, or is        —O—C(═O)—, or —HN—C(═O)—;    -   each R¹, R² and R³ is independently H or a non-interfering        substituent;    -   n is 0, 1 or 2;    -   Y is selected from the group consisting of    -   where m is 1 or 2; and    -   AA_(c) is an amino acid linked through a peptide bond to the        remainder of the compound.

The compounds are stated to be useful drugs for the selective treatmentof target tissues which contain compatible GST isoenzymes, andsimultaneously elevate the levels of GM progenitor cells in bone marrow.Disclosed embodiments for L include those that generate a drug that iscytotoxic to unwanted cells, including the phosphoramidate andphosphorodiamidate mustards.

One of the compounds has the formula

It is referred to in the patent as TER 286 and named asγ-glutamyl-α-amino-β-((2-ethyl-N,N,N,N-tetra(2′-chloro)ethylphosphoramidate)sulfonyl)propionyl-(R)-(−)-phenylglycine.This compound, later referred to as TLK286, has the CAS nameL-γ-glutamyl-3-[[2-[[bis[bis(2-chloroethyl)amino]phosphinyl]oxy]ethyl]sulfonyl]-L-alanyl-2-phenyl-(2R)-glycine.As the neutral compound, its proposed International Nonproprietary Nameis canfosfamide; and as its hydrochloride acid addition salt, its UnitedStates Adopted Name is canfosfamide hydrochloride. Canfosfamide and itssalts are anticancer compounds that are activated by the actions of GSTP1-1, and by GST A1-1, to release the cytotoxic phosphorodiamidatemustard moiety.

In vitro, canfosfamide has been shown to be more potent in the M6709human colon carcinoma cell line selected for resistance to doxorubicinand the MCF-7 human breast carcinoma cell line selected for resistanceto cyclophosphamide, both of which overexpress GST P1-1, over theirparental cell lines; and in murine xenografts of M7609 engineered tohave high, medium, and low levels of GST P1-1, the potency ofcanfosfamide hydrochloride was positively correlated with the level ofGST P1-1 (Morgan et al., Cancer Res., 58:2568 (1998)).

Canfosfamide hydrochloride is currently being evaluated in multipleclinical trials for the treatment of ovarian, breast, non-small celllung, and colorectal cancers. It has demonstrated significant singleagent antitumor activity and improvement in survival in patients withnon-small cell lung cancer and ovarian cancer, and single agentantitumor activity in colorectal and breast cancer. Evidence from invitro cell culture and tumor biopsies indicates that canfosfamide isnon-cross-resistant to platinum, paclitaxel, and doxorubicin (Rosario etal., Mol. Pharmacol, 58:167 (2000)), and also to gemcitabine. Patientstreated with canfosfamide hydrochloride show a very low incidence ofclinically significant hematological toxicity.

PCT Publication No. WO 95/09865 also discloses intermediates that arecompounds of the formula

and their amides, esters, and salts, where:

-   -   L is an electron withdrawing leaving group;    -   S⁺ is S or Se;    -   S* is —S(═O)—, —S(═O)₂—, —S(═NH)—, —S(═O)(═NH)—, —S⁺(C₁-C₆        alkyl)—, —Se(═O)—, —Se(═O)₂—, —Se(═NH)—, or —Se(═O)(═NH)—, or is        —O—C(═O)—, or —HN—C(═O)—;    -   each R¹, R² and R³ is independently H or a non-interfering        substituent;    -   n is 0, 1 or 2;    -   Y is selected from the group consisting of        where m is 1 or 2; and    -   AA_(c) is an amino acid linked through a peptide bond to the        remainder of the compound.

U.S. Pat. No. 6,506,739 [and PCT Publication No. WO 01/83496] disclosescompounds of the formula

where:

-   -   X is a halogen atom;    -   Q is O, S, or NH; and    -   R is hydrogen, optionally substituted lower alkyl, optionally        substituted aryl, or optionally substituted heteroaryl, or is        R′CO—, R′NHCO—, R′SO₂—, or R′NHSO₂— where R′ is hydrogen,        optionally substituted lower alkyl, optionally substituted aryl,        or optionally substituted heteroaryl; or R-Q together is        chlorine; and their salts.

The compounds are stated to be antitumor agents.

It would be desirable to develop other anticancer drugs having anefficacy and safety as good or better than canfosfamide and othercompounds of U.S. Pat. No. 5,556,942.

The disclosures of U.S. Pat. Nos. 5,556,942 and 6,506,739, and thedisclosures of other documents referred to in this application, areincorporated into this application by reference.

SUMMARY OF THE INVENTION

In a first aspect, this invention is compounds of formula A, B, and C:

where:

-   -   each R is independently hydrogen, C₁₋₆ alkyl, or —CH₂CH₂X, where        each X is independently Cl, Br, C₁₋₆ alkanesulfonyloxy,        halo-C₁₋₆ alkanesulfonyloxy, or benzenesulfonyloxy optionally        substituted with up to three substituents selected from halo,        C₁₋₃ alkyl, halo-C₁₋₃ alkyl, C₁₋₃ alkyloxy, or halo-C₁₋₃        alkyloxy, provided that at least two R's in each        phosphorodiamidate group are —CH₂CH₂X;    -   R¹ is optionally substituted alkyl, optionally substituted        heteroalkyl, optionally substituted aryl, optionally substituted        aralkyl, optionally substituted heteroaryl, or optionally        substituted heteroaralkyl; and    -   R² is optionally substituted alkanediyl, optionally substituted        heteroalkanediyl, optionally substituted arenediyl, optionally        substituted arenedialkyl, optionally substituted        heteroarenediyl, or optionally substituted heteroarenedialkyl,    -   and their salts.

In a second aspect, this invention is pharmaceutical compositionscomprising one or more compounds of the first aspect of this invention.

In a third aspect, this invention is methods of treating cancer by theadministration of a compound of the first aspect of this invention or apharmaceutical composition of the second aspect of this invention; aloneor in combination with other anticancer therapies.

In a fourth aspect, this invention is compounds of formula BB and CC:

where:

-   -   each R is independently hydrogen, C₁₋₆ alkyl or —CH₂CH₂X, where        each X is independently Cl, Br, C₁₋₆ alkanesulfonyloxy,        halo-C₁₋₆ alkanesulfonyloxy, or benzenesulfonyloxy optionally        substituted with up to three substituents selected from halo,        C₁₋₃ alkyl, halo-C₁₋₃ alkyl, C₁₋₃ alkyloxy, or halo-C₁₋₃        alkyloxy, provided that at least two R's in each        phosphorodiamidate group are —CH₂CH₂X;    -   R¹ is optionally substituted alkyl, optionally substituted        heteroalkyl, optionally substituted aryl, optionally substituted        aralkyl, optionally substituted heteroaryl, or optionally        substituted heteroaralkyl; and    -   R² is optionally substituted alkanediyl, optionally substituted        heteroalkanediyl, optionally substituted arenediyl, optionally        substituted arenedialkyl, optionally substituted        heteroarenediyl, or optionally substituted heteroarenedialkyl,    -   and their salts.

In a fifth aspect, this invention is pharmaceutical compositionscomprising one or more compounds of the fourth aspect of this invention.

In a sixth aspect, this invention is methods of treating cancer by theadministration of a compound of the fourth aspect of this invention or apharmaceutical composition of the fifth aspect of this invention; aloneor in combination with other anticancer therapies.

In a seventh aspect, this invention is methods of preparing compounds ofthe first and fourth aspects of this invention. Compounds of the firstaspect of this invention are conveniently prepared from compounds of thefourth aspect of this invention.

DETAILED DESCRIPTION OF THE INVENTION DEFINITIONS

“Alkyl” means a monovalent group derived from a saturated or unsaturated(but not aromatically unsaturated) C₁-C₁₀ hydrocarbon that may belinear, branched, or cyclic by removal of one hydrogen atom from acarbon atom. Examples are methyl, ethyl, propyl, 1-propenyl, isopropyl,butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl, cyclopentyl,cyclopenten-1-yl, cyclopropylmethyl, cyclohexyl, and cyclohexylmethyl.Saturated alkyls (including cycloalkyls) and C₁-C₆ alkyls are exemplary.Note that the definition of “alkyl” in this application is broader thanthe conventional definition and includes groups more commonly referredto as “cycloalkyl”, “cycloalkylalkyl”, “alkenyl”, and “alkynyl”.“Alkanediyl” means a divalent group derived from an “alkyl” (as thatterm is defined here) by removal of a second hydrogen atom from the samecarbon atom or another carbon atom, preferably from another carbon atom.Examples are 1,2-ethanediyl, but-2-en-1,4-diyl, 1,5-pentanediyl, and1,4-cyclohexanediyl.

A “substituted alkyl” is an alkyl substituted with up to three halogenatoms and/or up to three substituents selected from —CN, —NO₂, —OR, —SR,—COR, —OC(O)R, —C(O)OR, —NR₂, —NR₃ ⁺X, —PR₂, —PR₃ ⁺X, —SO₂OR, —OSO₂R,—SO₂NR₂, —NRSO₂R, —CONR₂, —NRCOR, and —NRC(O)OR, where each R is,independently, hydrogen, optionally R′-substituted alkyl, optionallyR′-substituted heteroalkyl, optionally R′-substituted aryl, optionallyR′-substituted heteroaryl, optionally R′-substituted aralkyl, oroptionally R′-substituted heteroaralkyl and each R′ is, independently, 1to 3 substituents selected from halo, —CN, —NO₂, —OH, C₁₋₃ alkyl, C₁₋₃alkyloxy, —SH, —NH₂, or —C(O)Oalkyl (preferably, 1 to 3 substituentsselected from halo, —CN, —NO₂, —OH, C₁₋₃ alkyl, C₁₋₃ alkyloxy, —SH, or—NH), or two R groups form a 4- or 5-member optionally R′-substitutedalkanediyl or optionally R′-substituted heteroalkanediyl, and X is ahalogen. Thus, for example, substituted alkyl groups include such groupsas trifluoromethyl, 3-chloropropyl, and 2-morpholinoethyl. Substitutedalkyl groups also include the residues (i.e. all except the thiol) ofthiol-amino acids such as cysteine, homocysteine, and penicillamine, andtheir esters and amides formed by reaction at the carboxy group andamides or sulfonamides formed by reaction at the amine group, such astheir N-protected forms and esters, where one substituent is —COOR andanother substituent is —NH₂ or —NRC(O)OR. “Substituted alkanediyl” meansalkanediyl substituted in the manner described above for an alkyl.Compounds of this invention also include compounds where anynon-aromatic amine having 1 or 2 hydrogen atoms present is protected byan amine-protecting group of the formula R*OC(O)— such astert-butoxycarbonyl, benzyloxycarbonyl, fluorenylmethoxycarbonyl, andother similar conventional carbamate-forming protecting groups. The term“substituted alkyl” specifically excludes dipeptides and higher peptidesbased on cysteine (i.e. where the “substituted alkyl” is bonded to thesulfonylethyl phosphorodiamidate portion of the molecule through thecysteine sulfur atom) and their esters, amides, and ester/amides.“Elaborated” refers to the conversion of a reactive substituent toanother typically more complex substituent, such as the conversion of anamine to an amide or sulfonamide, a carboxy group to an ester or amide,a hydroxy to an ester, and conversion of an amide or sulfonamide withone or more hydrogen atoms on the nitrogen to one where one or more ofthose hydrogen atoms is replaced by an optionally substituted alkyl,heteroalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl group.“Protected” has its conventional meaning in organic synthesis, namelythe temporary conversion of a reactive substituent to a substituent thatis non-reactive under the conditions of the reaction(s) proposed to becarried out; such as the protection of an amine as a carbamate asmentioned above.

“Heteroalkyl” means alkyl in which 1 to 3 of the carbon atoms arereplaced by O, S, or NR (where R is H or C₁₋₃ alkyl optionallysubstituted with halogen or hydroxy), including linear groups such as3-oxapentyl; monocyclic rings containing 5 or 6 ring atoms such as2-tetrahydrofuranyl, 2-pyrrolidinyl, 3-piperidinyl, 2-piperazinyl,4-methyl-1-piperazinyl, 4-dihydropyranyl, and 3-morpholinyl; and groupssuch as tetrahydrofuran-2-ylmethyl and piperidin-3-ylethyl.“Heteroalkanediyl” means a divalent group derived from a heteroalkyl byremoval of a second hydrogen atom, such as 3-oxapentane-1,5-diyl.Heteroalkyl and heteroalkanediyl groups also include those where a ringnitrogen is oxidized to form an N-oxide. A “cycloamino” group is acyclic heteroalkyl of 5 to 7 ring atoms containing a nitrogen ring atomby which the group is bonded to the remainder of the molecule of whichit forms a part and optionally containing a further ring heteroatomselected from O, S, and NR (where R is H or C₁₋₃ alkyl optionallysubstituted with halogen, hydroxy, or 1 or 2 phenyl groups).4-Methyl-1-piperazinyl, 4-(2-hydroxyethyl)-1-piperazinyl,4-(diphenylmethyl)-1-piperazinyl, and 4-morpholinyl are examples ofcycloamino groups. Compounds of this invention also include compoundswhere any —NR₂ group present is replaced by a cycloamino group.

“Substituted heteroalkyl” and “substituted hereteroalkanediyl” meanheteroalkyl and heteroalkanediyl substituted in the manner describedabove for substituted alkyl.

“Aryl” means a monovalent group derived from an aromatic hydrocarboncontaining 6 to 14 ring carbon atoms by removal of one hydrogen atomfrom a carbon atom, which is monocyclic (e.g., phenyl), condensedpolycyclic, for example, condensed bicyclic (e.g., naphthyl), or linkedpolycyclic, for example, linked bicyclic (e.g., biphenylyl). “Arenediyl”means a divalent group derived from an aryl by removal of a secondhydrogen atom from a carbon atom, such as 1,4-benzenediyl,1,5-naphthalenediyl, and biphenyl-4,4′-diyl. A preferred aryl is phenyl,and a preferred arenediyl is benzenediyl (any isomer).

“Substituted aryl” means aryl substituted with up to three substituentsselected from halo, —CN, —NO₂, —OR, optionally halo-substituted C₁₋₃alkyl, optionally halo-substituted C₁₋₃ alkyloxy, —SR, —COR, —OC(O)R,—C(O)OR, —NR₂, —NR₃ ⁺X, —PR₂, —PR₃ ⁺X—, —SO₂OR, —OSO₂R, —SO₂NR₂,—NRSO₂R, —CONR₂, —NRCOR, and —NRC(O)OR, where each R is hydrogen,optionally R′-substituted alkyl, optionally R′-substituted heteroalkyl,optionally R′-substituted aryl, optionally R′-substituted heteroaryl,optionally R′-substituted aralkyl, or optionally R′-substitutedheteroaralkyl (preferably, hydrogen or optionally R′-substituted alkyl)and each R′ is, independently, 1 to 3 substituents selected from halo,—CN, —NO₂, —OH, C₁₋₃ alkyl, C₁₋₃ alkyloxy, —SH, —NH₂, or —C(O)Oalkyl(preferably, halo, —CN, —NO₂, —OH, C₁₋₃ alkyl, C₁₋₃ alkyloxy, —SH, or—NH₂), or two R groups form a 4- or 5-member optionally R′-substitutedalkanediyl or optionally R′-substituted heteroalkanediyl, and X is ahalogen. Two adjacent substituents may also form a methylenedioxy orethylenedioxy group. Substituted aryl groups include aryl groupssubstituted with up to three substituents selected from the groupconsisting of halo, —CN, —NO₂, —OH, optionally halo-substituted C₁₋₃alkyl, optionally halo-substituted C₁₋₃ alkyloxy, —SH, and —NH₂, forexample, phenyl substituted in this way. “Substituted arenediyl” meansarenediyl substituted in the manner described above for an aryl.Preferred substituted aryls are substituted phenyls.

“Aralkyl” means alkyl substituted with aryl, such as benzyl andphenethyl. A preferred aralkyl is benzyl. “Arenedialkyl” means twoalkyls jointly substituted with arenediyl, such as benzene-1,4-dimethyl.A preferred arenedialkyl is benzenedimethyl (any isomer).

“Substituted aralkyl” means aralkyl in which one or both of the aryl andthe alkyl are substituted in the manner described above for substitutedaryl and substituted alkyl; and “substituted arenedialkyl” meansarenedialkyl in which one or more of the arenediyl and the two alkylsare substituted in the manner described above for substituted arenediyland substituted alkyl. The preferred substituted aralkyls aresubstituted benzyls; and preferred substituted arenedialkyls aresubstituted benzenedimethyls.

“Halogen” or “halo” means F, Cl, or Br.

“Heteroaryl” means aryl in which 1 to 4 (preferably 1 to 3) of the ringcarbon atoms are replaced by O, S, N, or NR (where R is H or C₁₋₃alkyl), preferably O, S, or NR, including monocyclic groups containing 5or 6 ring atoms such as furanyl, thienyl, pyrrolyl, oxazolyl,imidazolyl, pyridinyl, pyrazinyl, pyridazinyl, pyrimidinyl, and thelike, and bicyclic groups such as benzothiazolyl, purinyl, andbenzimidazolyl. Monocyclic rings are preferred. “Heteroarenediyl” meansa divalent group derived from a heteroaryl by removal of a secondhydrogen atom from a carbon atom. Heteroaryl and heteroatenediyl groupsalso include those where a ring nitrogen is oxidized to form an N-oxide.

“Substituted heteroaryl” and “substituted heteroatenediyl” meanheteroaryl and heteroarenediyl substituted in the manner described abovefor substituted aryl.

“Heteroaralkyl” means alkyl substituted with heteroaryl, such as2-thienylmethyl. “Heteroarenedialkyl” means two alkyls substitutedjointly with heteroarenediyl, such as 2,5-furanyldiethyl.

“Substituted heteroaralkyl” and “substituted heteroarenedialkyl” meanheteroaralkyl and heteroarenedialkyl substituted in the manner describedabove for substituted alkyl and substituted arenedialkyl.

“Salts” are described in the section entitled “Compounds of thisinvention”.

A “therapeutically effective amount” means that amount which, whenadministered to a human for treating a cancer, is sufficient to effecttreatment for the cancer. “Treating” or “treatment” of a cancer in ahuman includes one or more of:

-   -   (1) limiting/inhibiting growth of the cancer, i.e.,        limiting/arresting its development,    -   (2) reducing/preventing spread of the cancer, i.e.        reducing/preventing metastases,    -   (3) relieving the cancer, i.e., causing regression of the        cancer,    -   (4) reducing/preventing recurrence of the cancer, and    -   (5) palliating symptoms of the cancer.

“Combination therapy” means the administration of a compound of thefirst or fourth aspects of this invention and another anticancer therapyduring the course of cancer chemotherapy. Such combination therapy mayinvolve the administration of the compound of the first or fourth aspectof this invention before, during, and/or after the administration of theanother anticancer therapy. The administration of the compound of thefirst or fourth aspect of this invention may be separated in time fromthe administration of the another anticancer therapy by up to severalweeks, and may precede it or follow it, but more commonly theadministration of the compound of the first or fourth aspect of thisinvention will accompany at least one aspect of the another anticancertherapy (such as the administration of one dose of a chemotherapeuticagent, molecular targeted therapy agent, biologic therapy agent, orradiation therapy) within up to 48 hours, and most commonly within lessthan 24 hours.

“Another anticancer therapy” is an anticancer therapy that is not atreatment with a compound of the first or fourth aspect of thisinvention. Such “another anticancer therapies” include chemotherapy;molecular targeted therapy; biologic therapy; and radiotherapy. Thesetherapies are those used as monotherapy or in combination therapy.

Chemotherapeutic agents include:

-   -   alkylating agents, including:    -   alkyl sulfonates such as busulfan,    -   ethyleneimine derivatives such as thiotepa,    -   nitrogen mustards such as chlorambucil, cyclophosphamide,        estramustine, ifosfamide, mechlorethamine,    -   melphalan, and uramustine,    -   nitrosoureas such as carmustine, lomustine, and streptozocin,    -   triazenes such as dacarbazine, procarbazine, and temozolamide,        and    -   platinum compounds such as cisplatin, carboplatin, oxaliplatin,        satraplatin, and picoplatin;    -   antimetabolites, including:    -   antifolates such as methotrexate, permetrexed, raltitrexed, and        trimetrexate,    -   purine analogs such as cladribine, chlorodeoxyadenosine,        clofarabine, fludarabine, mercaptopurine,    -   pentostatin, and thioguanine,    -   pyrimidine analogs such as azacitidine, capecitabine,        cytarabine, edatrexate, floxuridine, fluorouracil,    -   gemcitabine, and troxacitabine;    -   natural products, including:    -   antitumor antibiotics such as bleomycin, dactinomycin,        mithramycin, mitomycin, mitoxantrone,    -   porfiromycin, and anthracyclines such as daunorubicin (including        liposomal daunorubicin), doxorubicin (including liposomal        doxorubicin), epirubicin, idarubicin, and valrubicin,    -   enzymes such as L-asparaginase and PEG-L-asparaginase,    -   microtubule polymer stabilizers such as the taxanes paclitaxel        and docetaxel,    -   mitotic inhibitors such as the vinca alkaloids vinblastine,        vincristine, vindesine, and vinorelbine,    -   topisomerase I inhibitors such as the camptothecins irinotecan        and topotecan, and    -   topoisomerase II inhibitors such as amsacrine, etoposide, and        teniposide;    -   hormones and hormone antagonists, including:    -   androgens such as fluoxymesterone and testolactone,    -   antiandrogens such as bicalutamide, cyproterone, flutamide, and        nilutamide,    -   aromatase inhibitors such as arninoglutethimide, anastrozole,        exemestane, formestane, and letrozole,    -   corticosteroids such as dexamethasone and prednisone,    -   estrogens such as diethylstilbestrol,    -   antiestrogens such as fulvestrant, raloxifene, tamoxifen, and        toremifine,    -   LHRH agonists and antagonists such as buserelin, goserelin,        leuprolide, and triptorelin,    -   progestins such as medroxyprogesterone acetate and megestrol        acetate, and    -   thyroid hormones such as levothyroxine and liothyronine; and    -   miscellaneous agents, including altretamine, arsenic trioxide,        gallium nitrate, hydroxyurea, levamisole,    -   mitotane, octreotide, procarbazine, suramin, thalidomide,        lenalidomide, photodynamic compounds such as methoxsalen and        sodium porfimer, and proteasome inhibitors such as bortezomib.

Molecular targeted therapy agents include:

-   -   functional therapeutic agents, including:    -   gene therapy agents,    -   antisense therapy agents,    -   tyrosine kinase inhibitors such as erlotinib hydrochloride,        gefitinib, imatinib mesylate, and semaxanib, and    -   gene expression modulators such as the retinoids and rexinoids,        e.g. adapalene, bexarotene, trans-retinoic acid, 9-cis-retinoic        acid, and N-(4-hydroxyphenyl)retinamide;    -   phenotype-directed therapy agents, including:    -   monoclonal antibodies such as alemtuzumab, bevacizumab,        cetuximab, ibritumomab tiuxetan,    -   rituximab, and trastuzumab,    -   immunotoxins such as gemtuzumab ozogamicin,    -   radioimmunoconjugates such as ¹³¹I-tositumomab, and    -   cancer vaccines.

Biologic therapy agents include:

-   -   interferons such as interferon-α_(2a) and interferon-α_(2b), and    -   interleukins such as aldesleukin, denileukin diftitox, and        oprelvekin.

In addition to these agents intended to act against cancer cells,anticancer therapies include the use of protective or adjunctive agents,including:

-   -   cytoprotective agents such as amifostine, dexrazoxane, and        mesna,    -   phosphonates such as pamidronate and zoledronic acid, and    -   stimulating factors such as epoetin, darbeopetin, filgrastim,        PEG-filgrastim, and sargramostim.

Combination cancer therapy regimens with which the compounds of thefirst aspect of this invention may be combined include all regimensinvolving the use of two or more of the anticancer therapies (anticanceragents) such as those mentioned in paragraphs [0038] to [0040] aboveand/or radiotherapy, optionally including protective and adjunctiveagents such as those mentioned in paragraph [0041] above; and thecompound of the first or second aspect of this invention can be added toexisting anticancer regimens known for the treatment of various cancers,such as the regimens mentioned in such books as Cancer Chemotherapy andBiotherapy: Princples and Practice, 3rd ed. (2001), Chabner and Longo,eds., and Handbook of Cancer Chemotherapy, 6th ed. (2003), Skeel, ed.,both from Lippincott Williams & Wilkins, Philadelphia, Penn., U.S.A.;and regimens for anticancer therapies, especially chemotherapies, may befound on Web sites such as those maintained by the National CancerInstitute (www.cancer.gov), the American Society for Clinical Oncology(www.asco.org), and the National Comprehensive Cancer Network(www.nccn.org).

Many combination chemotherapeutic regimens are known to the art, such ascombinations of platinum compounds and taxanes, e.g.carboplatin/paclitaxel, capecitabine/docetaxel, the “Cooper regimen”,fluorouracil-levamisole, fluorouracil-leucovorin,methotrexate-leucovorin, and those known by the acronyms ABDIC, ABVD,AC, ADIC, AI, BACOD, BACOP, BVCPP, CABO, CAD, CAE, CAF, CAP, CD, CEC,CF, CHOP, CHOP+rituximab, CIC, CMF, CMFP, CyADIC, CyVADIC, DAC, DVD,FAC, FAC-S, FAM-S, FOLFOX-4, FOLFOX-6, M-BACOD, MACOB-B, MAID, MOPP,MVAC, PCV, T-5, VAC, VAD, VAPA, VAP-Cyclo, VAP-II, VBM, VBMCP, VIP, VP,and the like.

Combinations of chemotherapies and molecular targeted therapies,biologic therapies, and radiation therapies are also well known to theart; including therapies such as trastuzumab+paclitaxel, alone or infurther combination with carboplatin, for certain breast cancers, andmany other such regimens for other cancers; and the “Dublin regimen” and“Michigan regimen”, both for esophageal cancer, and many other suchregimens for other cancers.

“Comprising” or “containing” and their grammatical variants are words ofinclusion and not of limitation and mean to specify the presence ofstated components, groups, steps, and the like but not to exclude thepresence or addition of other components, groups, steps, and the like.Thus “comprising” does not mean “consisting of”, “consistingsubstantially of”, or “consisting only of”; and, for example, aformulation “comprising” a compound must contain that compound but mayalso may contain other active ingredients and/or excipients.

Compounds of this Invention

In a first aspect, this invention is sulfonylethyl phosphorodiamidates,compounds of formula A, B, and C:

where:

-   -   each R is independently hydrogen, C₁₋₆ alkyl, or —CH₂CH₂X, where        each X is independently Cl, Br, C₁₋₆ alkanesulfonyloxy,        halo-C₁₋₆ alkanesulfonyloxy, or benzenesulfonyloxy optionally        substituted with up to three substituents selected from halo,        C₁₋₃ alkyl, halo-C₁₋₃ alkyl, C₁₋₃ alkyloxy, or halo-C₁₋₃        alkyloxy, provided that at least two R's in each        phosphorodiamidate group are —CH₂CH₂X;    -   R¹ is optionally substituted alkyl, optionally substituted        heteroalkyl, optionally substituted aryl, optionally substituted        aralkyl, optionally substituted heteroaryl, or optionally        substituted heteroaralkyl; and    -   R² is optionally substituted alkanediyl, optionally substituted        heteroalkanediyl, optionally substituted arenediyl, optionally        substituted arenedialkyl, optionally substituted        heteroarenediyl, or optionally substituted heteroarenedialkyl,    -   and their salts.

In a fourth aspect, this invention is thioethyl phosphorodiamidates,compounds of formula BB and CC:

where:

-   -   each R is independently hydrogen, C₁₋₆ alkyl, or —CH₂CH₂X, where        each X is independently Cl, Br, C₁₋₆ alkanesulfonyloxy,        halo-C₁₋₆ alkanesulfonyloxy, or benzenesulfonyloxy optionally        substituted with up to three substituents selected from halo,        C₁₋₃ alkyl, halo-C₁₋₃ alkyl, C₁₋₃ alkyloxy, or halo-C₁₋₃        alkyloxy, provided that at least two R's in each        phosphorodiamidate group are —CH₂CH₂X;    -   R¹ is optionally substituted alkyl, optionally substituted        heteroalkyl, optionally substituted aryl, optionally substituted        aralkyl, optionally substituted heteroaryl, or optionally        substituted heteroaralkyl; and    -   R² is optionally substituted alkanediyl, optionally substituted        heteroalkanediyl, optionally substituted arenediyl, optionally        substituted arenedialkyl, optionally substituted        heteroarenediyl, or optionally substituted heteroarenedialkyl,    -   and their salts.

Salts (for example, pharmaceutically acceptable salts) of the compoundsof formulae A, B, C, BB, and CC are included in the present inventionand are useful in the compositions, methods, and uses described in thisapplication (see Berge et al., J. Pharm. Sci., 66:1 (1971) for anonexclusive list of pharmaceutically acceptable salts).

These salts include salts that may be formed when acidic protons presentare capable of reacting with inorganic or organic bases. Typically theparent compound is treated with an excess of an alkaline reagent, suchas hydroxide, carbonate or alkoxide, containing an appropriate cation.Cations such as Na⁺, K⁺, Ca²⁺, Mg²⁺ and NH₄₊ are examples of cationspresent in pharmaceutically acceptable salts. Suitable inorganic bases,therefore, include calcium hydroxide, potassium hydroxide, sodiumcarbonate and sodium hydroxide. Salts may also be prepared using organicbases, such as salts of primary, secondary and tertiary amines,substituted amines including naturally-occurring substituted amines, andcyclic amines including isopropylamine, trimethylamine, diethylamine,triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol,tromethamine, lysine, arginine, histidine, caffeine, procaine,hydrabamine, choline, betaine, ethylenediamine, glucosamine,N-alkylglucamines, theobromine, purines, piperazine, piperidine,N-ethylpiperidine, and the like.

If a compound of the first or fourth aspects of this invention containsa basic group, such as an amino or phosphino group, it may be preparedas an acid addition salt. Acid addition salts of the compounds areprepared in a standard manner in a suitable solvent from the parentcompound and an excess of an acid, such as hydrochloric acid,hydrobromic acid, sulfuric acid (giving the sulfate and bisulfatesalts), nitric acid, phosphoric acid and the like, and organic acidssuch as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalicacid, malic acid, malonic acid, succinic acid, maleic acid, fumaricacid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelicacid, methanesulfonic acid, ethanesulfonic acid, salicylic acid,4-toluenesulfonic acid, hexanoic acid, heptanoic acid,cyclopentanepropionic acid, lactic acid, 2-(4-hydroxybenzoyl)benzoicacid, 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid,benzenesulfonic acid, 4-chlorobenzenesulfonic acid,2-naphthalenesulfonic acid, camphorsulfonic acid,4-methylbicyclo[2.2.2.]oct-2-ene-1-carboxylic acid, glucoheptonic acid,gluconic acid, 4,4′-methylenebis(3-hydroxy-2-naphthoic)acid,3-phenylpropionic acid, trimethylacetic acid, tea-butylacetic acid,laurylsulfuric acid, glucuronic acid, glutamic acid,3-hydroxy-2-naphthoic acid, stearic acid, muconic acid and the like.

Certain of the compounds may form inner salts or zwitterions.

Certain compounds of formula A, B, and BB of the invention may containone or more chiral centers (for example, those based on amino acids). Insuch cases, all individual stereoisomers and racemic and non-racemicmixtures of the stereoisomers of the compounds (and their salts) areincluded in this invention. Typically the stereochemistry of thecompounds of this invention will be dictated by the stereochemistry ofthe thiols of formula A¹ and B¹, and methods for the isolation ofindividual stereoisomers of such thiols, for example, the resolution ofthiol-amino acids such as cysteine, are well known to a person ofordinary skill in the art and are therefore not described here. In manyinstances, such as with cysteine and penicillamine, individualstereoisomers and racemic mixtures of stereoisomers are commerciallyavailable; and other thiols such as dithiothreitol and dithioetythritolexist as individual stereoisomers.

The compounds are named in this application semi-systematically based onthe CAS name of canfosfamide (i.e. with the thioethyl or sulfonylethylphosphorodiamidate portion of the molecule named as a substituent on theremainder of the molecule). Thus, the compounds 70A, 128A, 23B, and 1Cbelow:

are named3-[[2-[[bis[bis(2-chlotoethyl)amino]phosphinyl]oxy]ethyl]sulfonyl]-L-alanine,α-[[2-[[bis[bis(2-chloroethyl)amino]phosphinyl]oxy]ethyl]sulfonyl]toluene,1,5-di[[2-[[bis[bis(2-chlotoethyl)amino]phosphinyl]oxy]ethyl]sulfonyl]naphthalene,and di[2-[[bis[bis(2-chlotoethyl)amino]phosphinyl]oxy]ethyl]sulfone,respectively.

Compounds of this invention include those compounds of formulae A, B, C,BB, and CC where one or more of the following is true:

-   -   1. each R is —CH₂CH₂X;    -   2. each X is Cl, Br, methanesulfonyloxy,        trifluotomethanesulfonyloxy, benzenesulfonyloxy, or        4-toluenesulfonyloxy; especially where X is Cl or Br;        particularly Cl;    -   3. the compound is a compound of formula A;    -   4. R¹ is substituted with one or mote groups that enhance the        solubility of the compound over a compound that is not so        substituted, for example, hydroxy, catboxy, sulfo, amino,        quaternary ammonium, phosphino, and quaternary phosphonium;    -   5. R¹ is optionally substituted alkyl, or optionally substituted        aryl, or optionally substituted aralkyl; and    -   6. R¹ is the residue of an optionally protected thiol-amino        acid, or an elaborated thiol-amino acid such as an amide or        ester formed by reaction at the carboxy group and/or amide or        sulfonamide formed by reaction at the amine group, preferably        the residue of a thiol-amino acid;    -   7. R¹ is the residue of 2-mercaptoacetic acid or        3-mercaptopropionic acid, or an elaborated acid such as an amide        or ester formed by reaction at the carboxy group, particularly        the residue of an optionally elaborated 2-mercaptoacetamide;    -   8. the compound is a compound of formula B;    -   9. R² is substituted with one or more groups that enhance the        solubility of the compound over a compound that is not so        substituted, for example, hydroxy, carboxy, sulfo, amino,        quaternary ammonium, phosphino, and quaternary phosphonium; or    -   10. the compound is a compound of formula C.

Compounds of this invention include each of the compounds described inthe specification and claims of this application as filed, including inthe Examples below, especially compounds 5A, 13A, 15A, 23A, 70A, 128A,171A, 172A, and 180A, and their salts. Compositions and methods of thisinvention include compositions and methods where the compound is one ofthose mentioned in the preceding sentence.

Preparation of the Compounds

Sulfonylethyl phosphorodiamidates of formula A, B, and C mayconveniently be prepared by oxidation of the corresponding thioethylphosphorodiamidates of formulae AA, BB, and CC (with any reactive moietyin R¹ or R² protected against oxidation if necessary), as follows:

This oxidation may be performed by any of the methods known in the artfor the oxidation of thioethers to sulfones, such as the use of peracids(peroxycarboxylic acids), persulfates, perborates, peroxides, ozone,iodosyl reagents, halogens, and the like. If the compound of formula AAor BB contains a reactive moiety in R¹ or R², such as an amine group,that reactive moiety may be protected before the oxidation and theresulting protected compound of formula A or B deprotected if desired toyield the final compound (though the protected compounds are also amongcompounds of this invention). Such protection and deprotection is seenin Synthetic Example 2, for example. Where a peracid is used, a typicalprocedure involves dissolving the compound of formula AA, BB, or CC in asolvent such as acetic acid or isopropyl acetate at reduced temperature,followed by the addition of the peracid (e.g. peracetic acid) in excess.

After oxidation and optional deprotection, the compounds of formula Aand formula B may be elaborated by synthetic methods known per se, asdiscussed later in this application.

The thioethyl phosphorodiamidates of formula AA and BB may convenientlybe prepared by one of two methods:

-   -   (1) reaction of the corresponding thiols of formula A¹ and B¹        with a 2-X¹-ethyl phosphorodiamidate (where X¹ is Cl, Br, or an        alkane- or arenesulfonyloxy group such as methanesulfonyloxy,        benzenesulfonyloxy, or 4-toluenesulfonyloxy), as follows:    -   (2) (a) reaction of the thiols of formula A¹ and B¹ with a        2-X¹-ethanol (where X¹ is Cl, Br, C₁₋₆ alkanesulfonyloxy,        halo-C₁₋₆ alkanesulfonyloxy, or benzenesulfonyloxy optionally        substituted with up to three substituents selected from halo,        C₁₋₃ alkyl, halo-C₁₋₃ alkyl, C₁₋₃ alkyloxy, or halo-C₁₋₃        alkyloxy) to first form the corresponding 2-hydroxyethyl thiols        of formula A² and B², followed by (b) reaction of the        2-hydroxyethyl thiols with a phosphorodiamidyl halide or alkane-        or arenesulfonate (X² is Cl, Br, C₁₋₆ alkanesulfonyloxy,        halo-C₁₋₆ alkanesulfonyloxy, or benzenesulfonyloxy optionally        substituted with up to three substituents selected from halo,        C₁₋₃ alkyl, halo-C₁₋₃ alkyl, C₁₋₃ alkyloxy, or halo-C₁₋₃        alkyloxy), as follows:

The direct conversion of thiols of formula A¹ and B¹ to the thioethylphosphorodiamidates of formula AA and BB may be performed by any of themethods known in the art for alkylation of thiols. A typical procedureinvolves dissolving the thiol of formula A¹ or B¹ in a polar solventsuch as water, an alcohol, dimethylformamide, or tetrahydrofuran, thentreating it with a base such as a hydroxide, alkoxide, fluoride, ortertiary amine or amide base to form the thiolate anion, followed byadding the phosphorodiamidate. Thiolate displacement of the leavinggroup X¹ of the 2-X¹-ethyl phosphorodiamidate gives the compounds offormula AA and BB.

Alternatively, the thiols of formula A¹ and B¹ may be treated withethanol 2-substituted with Cl, Br, C₁₋₆ alkanesulfonyloxy, halo-C₁₋₆alkanesulfonyloxy, or benzenesulfonyloxy optionally substituted with upto three substituents selected from halo, C₁₋₃ alkyl, halo-C₁₋₃ alkyl,C₁₋₃ alkyloxy, or halo-C₁₋₃ alkyloxy, e.g. 2-chloroethanol, under theconditions of the previous paragraph to give the 2-hydroxyethylthiolcompounds of formula A² and B², which may then be converted to thecompounds of formula AA and BB, typically by reacting the compounds offormula A² and B² with a phosphorodiamidyl halide or alkane- orarenesulfonate in an aprotic solvent such as tetrahydrofuran, toluene,or dichloromethane in the presence of an base such as an alkoxide ortertiary amine.

After optional deprotection, the compounds of formula AA and formula BBmay be elaborated by synthetic methods known per se, as discussed laterin this application.

The compounds of formula CC are conveniently prepared by a two-stepsynthesis, in which 2,2′-thiodiethanol is reacted with asub-stoichiometric amount of a phosphorodiamidyl halide or alkane- orarenesulfonate in an aprotic solvent in the presence of a base such asan alkoxide to give the mono-phosphorodiamidate ester, and that ester isthen reacted with an excess of the phosphorodiamidyl halide or alkane-or arenesulfonate, again in an aprotic solvent in the presence of a basesuch as an alkoxide, to give the bis-phosphorodiamidate ester, asfollows:

Pharmaceutical Compositions and Administration

The second and fifth aspects of this invention are pharmaceuticalcompositions comprising a compound of the first or fourth aspect of thisinvention and optionally a pharmaceutically acceptable excipient.

The compounds of the first and fourth aspects of this invention may beadministered by any route suitable to the subject being treated and thenature of the subject's condition. Routes of administration includeadministration by injection, including intravenous, intraperitoneal,intramuscular, and subcutaneous injection, by transmucosal ortransdermal delivery, through topical applications, nasal spray,suppository and the like or may be administered orally. Formulations mayoptionally be liposomal formulations, emulsions, formulations designedto administer the drug across mucosal membranes or transdermalformulations. Suitable formulations for each of these methods ofadministration may be found, for example, in Remington: The Science andPractice of Pharmacy, 20th ed., A. Gennaro, ed., Lippincott Williams &Wilkins, Philadelphia, Penn., U.S.A. Typical formulations will be eitheroral or solutions for intravenous infusion. Typical dosage forms will betablets or capsules for oral administration, solutions for intravenousinfusion, and lyophilized powders for reconstitution as solutions forintravenous infusion.

Depending on the intended mode of administration, the pharmaceuticalcompositions may be in the form of solid, semi-solid or liquid dosageforms, preferably in unit dosage form suitable for single administrationof a precise dosage. In addition to an effective amount of the activeingredient(s), the compositions may contain suitablepharmaceutically-acceptable excipients, including adjuvants whichfacilitate processing of the active compounds into preparations whichcan be used pharmaceutically. “Pharmaceutically acceptable excipient”refers to an excipient or mixture of excipients which does not interferewith the effectiveness of the biological activity of the activeingredient(s) and which is not toxic to the host to which it isadministered.

For solid compositions, conventional excipients include, for example,pharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharin, talc, cellulose, glucose, sucrose, magnesiumcarbonate, and the like. Liquid pharmacologically administrablecompositions can, for example, be prepared by dissolving, dispersing,etc., an active compound as described herein and optional pharmaceuticaladjuvants in an excipient, such as, for example, water, saline, aqueousdextrose, glycerol, ethanol, and the like, to thereby form a solution orsuspension. If desired, the pharmaceutical composition to beadministered may also contain minor amounts of nontoxic auxiliaryexcipients such as wetting or emulsifying agents, pH buffering agentsand the like, for example, sodium acetate, sorbitan monolaurate,triethanolamine sodium acetate, triethanolamine oleate, etc.

For oral administration, the composition will generally take the form ofa tablet or capsule, or it may be an aqueous or nonaqueous solution,suspension or syrup. Tablets and capsules are preferred oraladministration forms. Tablets and capsules for oral use will generallyinclude one or more commonly used excipients such as lactose and cornstarch. Lubricating agents, such as magnesium stearate, are alsotypically added. When liquid suspensions are used, the active agent maybe combined with emulsifying and suspending excipients. If desired,flavoring, coloring and/or sweetening agents may be added as well. Otheroptional excipients for incorporation into an oral formulation includepreservatives, suspending agents, thickening agents, and the like.

Injectable formulations can be prepared in conventional forms, either asliquid solutions or suspensions, solid forms suitable for solubilizationor suspension in liquid prior to injection, or as emulsions or liposomalformulations. The sterile injectable formulation may also be a sterileinjectable solution or a suspension in a nontoxic parenterallyacceptable diluent or solvent. Among the acceptable vehicles andsolvents that may be employed are water, Ringer's solution and isotonicsodium chloride solution. In addition, sterile, fixed oils, fatty estersor polyols are conventionally employed as solvents or suspending media.

The pharmaceutical compositions of this invention may also be formulatedas lyophilized powders for parenteral administration. Powders may bereconstituted by addition of water or other aqueous medium and thenfurther diluted with a suitable diluent prior to use. The liquidformulation is generally a buffered, isotonic, aqueous solution.Examples of suitable diluents are isotonic saline solution, 5% dextrosein water, and buffered sodium or ammonium acetate solution.Pharmaceutically acceptable solid or liquid excipients may be added toenhance or stabilize the composition, or to facilitate preparation ofthe composition.

Typically, a pharmaceutical composition of the present invention ispackaged in a container with a label, or instructions, or both,indicating use of the pharmaceutical composition in the treatment ofcancer.

The pharmaceutical composition may additionally contain one or moreother pharmacologically active agents in addition to a compound of thisinvention. These additional active agents will typically be useful intreating cancer, or for enhancing the treatment of cancer by compoundsof this invention.

Methods of Using the Compounds

The compounds of the first and fourth aspect of this invention haveactivity against human cancer cell lines, as demonstrated in the invitro and in vivo Examples below, and are therefore considered to beuseful as human cancer chemotherapeutic agents, for the treatment ofhuman cancers.

Thus, the third and sixth aspects of this invention include methods oftreating cancer in humans by administering a therapeutically effectiveamount of a compound of the first or fourth aspect of this invention, ora pharmaceutical composition of the second or fifth aspect of thisinvention, to the human. Optionally, the methods further comprisetreating the human with another anticancer therapy, such as a therapyalready conventional for the cancer being treated.

Cancers that are particularly treatable by the method of this inventionare cancers with sensitivity to inducers of apoptosis, and morespecifically those cancers that express or, particularly, overexpressone or more glutathione S-transferase isoenzymes. Cancers that expressor overexpress one or more glutathione S-transferase isoenzymes whentreated with other anticancer compounds or combination cancerchemotherapy regimens are especially treatable by the method of thisinvention. Such cancers include cancers of the brain, breast, bladder,cervix, colon and rectum, esophagus, head and neck, kidney, lung, liver,ovary, pancreas, prostate, and stomach; leukemias such as ALL, AML,AMML, CLL, CML, CMML, and hairy cell leukemia; Hodgkin's andnon-Hodgkin's lymphomas; mesotheliomas, multiple myeloma; and sarcomasof bone and soft tissue. Cancers particularly treatable by the method ofthis invention include breast, ovarian, colorectal, and non-small celllung cancers.

The amount of the compound of the first or fourth aspect of thisinvention that is administered to the human (either alone or, moreusually, in a composition of the second or fifth aspect of thisinvention) should be a therapeutically effective amount when used aloneor when used in conjunction with the another anticancer therapy (if thecompound of the first or fourth aspect of this invention is administeredin conjunction with another anticancer therapy); and similarly theamount of the another anticancer therapy that is administered to themammal (if the compound of the first or fourth aspect of this inventionis administered in conjunction with another anticancer therapy) shouldbe a therapeutically effective amount when used in conjunction with thecompound of the first or fourth aspect of this invention. However, thetherapeutically effective amount of either the compound of the first orfourth aspect of this invention and the amount of the another anticancertherapy when administered in combination cancer chemotherapy may each beless than the amount which would be therapeutically effective ifdelivered to the human alone. It is common in cancer therapy, though, touse the maximum tolerated dose of the or each therapy, with a reductiononly because of common toxicity of the therapies used or potentiation ofthe toxicity of one therapy by another. Because of the lack ofcross-resistance of canfosfamide, for example, with several commonchemotherapeutic agents, and its relative lack of clinically severetoxicity, especially its lack of clinically severe hematologicaltoxicity, it is expected that compounds of the first and fourth aspectof this invention will be administrable at essentially their maximumtolerated dose as a single agent, and no reduction in the amount of theanother anticancer therapy will be required.

The compounds of the first and fourth aspects of this invention, orpharmaceutical compositions of the second and fifth aspects of thisinvention, are thus used to treat cancer in humans requiring suchtreatment, by administering a therapeutically effective amount of thechosen compound or composition. Therapeutically effective amounts ofcompounds of the invention are in the range of 10-10,000 mg/m², forexample, 30-3000 mg/m² or 100-1000 mg/m². Dosing may be at 1-35 dayintervals; for example, about 500-1000 mg/m² at 1-5 week intervals,especially at 1, 2, 3, or 4 week intervals, or at higher frequenciesincluding as frequently as once/day for several (e.g. 5 or 7) days, withthe dosing repeated every 2, 3, or 4 weeks, or constant infusion for aperiod of 6-72 hours, also with the dosing repeated every 2, 3, or 4weeks. Suitable dosages and dose frequencies will be readilydeterminable by a person of ordinary skill in the art having regard tothat skill and this disclosure. No unacceptable toxicological effectsare expected when compounds of the invention are administered inaccordance with the present invention.

Suitable dosing for the other anticancer therapy (if the compound of thefirst or fourth aspect of this invention is used in combination) will bethe dosing already established for that therapy, as described in suchdocuments as those listed in paragraph [0042]. Such dosing varies widelywith the therapy: for example, capecitabine (2500 mg/m² orally) is dosedtwice daily for 2 weeks on and 1 week off, imatinib mesylate (400 or 600mg/day orally) is dosed daily, rituximab is dosed weekly, paclitaxel(135-175 mg/me and docetaxel (60-100 mg/m) are dosed weekly to everythree weeks, carboplatin (4-6 mg/mL*min) is dosed once every 3 or 4weeks (though the doses may be split and administered over severaldays), nitrosourea alkylating agents such as carmustine are dosed asinfrequently as once every 6 weeks. Radiotherapy may be administered asfrequently as weekly (or even within that split into smaller dosagesadministered daily).

A person of ordinary skill in the art of cancer therapy will be able toascertain a therapeutically effective amount of the compound of thefirst or second aspect of this invention and a therapeutically effectiveamount of another anticancer therapy for a given cancer and stage ofdisease without undue experimentation and in reliance upon personalknowledge and the disclosure of this application.

Combination therapies include the combination administration of acompound of the first aspect of this invention with a platinum compoundsuch as carboplatin or cisplatin, optionally in further combination withgemcitabine or a taxane such as docetaxel or paclitaxel; withgemcitabine; with a taxane; with an anthracycline such as doxorubicin orliposomal doxorubicin; with oxaliplatin, optionally in furthercombination with capecitabine or fluorouracil/leucovorin; and withgemcitabine or a platinum compound such as carboplatin or cisplatin, infurther combination with a vinca alkaloid such as vinorelbine.

EXAMPLES

The following examples illustrate the preparation of compounds of thisinvention, and their activity in predictive in vitro and in vivoanticancer assays.

Synthetic Examples

The compounds of this invention are prepared by conventional methods oforganic chemistry. See, for example, Larock, “Comprehensive OrganicTransformations”, Wiley-VCH, New York, New York, U.S.A. In some cases,protective groups may be introduced and later removed. Suitableprotective groups for amino, hydroxyl, and carboxyl groups are describedin Greene et al. “Protective Groups in Organic Synthesis”, 2nd ed.,1991, John Wiley and Sons, New York, New York, U.S.A. The compounds ofthis invention can be synthesized, generally following the syntheticschemes illustrated earlier in this application, as shown in thefollowing examples or by modifying the exemplified syntheses by meansknown to those of ordinary skill in the art.

The synthetic examples below show compounds where R in formulae A, B, C,BB, and CC are all 2-chloroethyl. It will be apparent that compoundswhere R is other than 2-chloroethyl may be produced by parallel methods:for example:

-   -   (a) compounds where R is 2-bromoethyl may be made by reacting        bis(2-bromoethyl)amine, POCl₃, and ethylene glycol to produce        2-hydroxyethyl tetrakis(2-bromoethyl)phosphorodiamidate,        esterifying to an appropriate sulfonate ester, and using that to        alkylate the thiols of formula A¹ and B¹ or 2,2′-thiodiethanol        to form the compounds of formulae AA, BB, and CC;    -   (b) compounds where R is 2-(Z-sulfonyloxy)ethyl may be made by        using bis(2-hydroxyethylamine) instead of bis(2-bromoethylamine)        in the process of (a) above, then forming the sulfonate esters;        and    -   (c) compounds where the R groups are not identical (e.g. where        one R on each amine is 2-chloroethyl and the other is hydrogen)        may be made by using the appropriate amine (e.g.        2-chloroethylamine) in the process of (a) above.

A person of ordinary skill in the art will have no difficulty, havingregard to that skill and this disclosure, in preparing compounds where Ris other than 2-chloroethyl.

It will also be apparent that when the R¹ group of formula A or the R²group of formula B contain reactive (e.g. amino, carboxy) groups, thatthese groups may be elaborated to produce other compounds. Thiselaboration of the R¹ and R² groups may take place at the thiol stage(elaboration of the R¹ group of formula A¹ or of the R² group of formulaB¹), or at the thioethyl phosphorodiamidate stage (elaboration of the R¹group of formula AA or of the R² group of formula BB), or at thesulfonylethyl phosphorodiamidate stage (elaboration of the R¹ group offormula A or of the R² group of formula B). All such elaborations may beperformed by synthetic methods known per se. A person of ordinary skillin the art will have no difficulty, having regard to that skill and thisdisclosure, in elaborating R¹ and R² groups from those readily availableto prepare the full range of the compounds of this invention.

For example, considering elaborated analogs of compound 13A (thepreparation of which is illustrated in Synthetic Example 1 below):2-mercaptoacetic acid may be alkylated with a phosphorodiamidate such as2-(4-bromobenzenesulfonyloxy)ethyltetrakis(2-chloroethyl)-phosphorodiamidate to give compounds such as2-[[2-[[bis[bis(2-chloroethyl)amino]phosphinyl]oxy]-ethyl]thio]aceticacid. This may be oxidized to2-[[2-[[bis[bis(2-chloroethyl)amino]phosphinyl]oxy]-ethyl]sulfonyl]aceticacid, compound 14A. The acid may, either before or after oxidation, bereacted with an amine in the presence of a base and coupling reagent, toform an amide. Compound 13A could be made this way, though it may bedirectly prepared as described below, and compounds 18A to 24A areexamples of compounds that may be made this way. Similar compoundshaving longer carbon chains between the carboxy group and the sulfonylgroup, e.g. compound 29A and derivatives such as compounds 53A to 59A,may be prepared by starting with 3-mercaptopropionic acid and similarthio-acids. Esters may similarly be prepared by esterification.Conversely, starting with a mercaptoalkylamine (temporarily protected atthe amine), alkylating the sulfur atom, oxidizing, and deprotecting,allows the preparation of reverse amides. Also, starting with2-mercaptoethanol and alkylating and oxidizing allows the preparation ofcompound 16A, and the hydroxy group can be esterified to give reverseesters. Similar elaborations can be made from compounds such as 139A(allowing the preparation of compounds such as compounds 154A to 178A)and 141A (allowing the preparation of compounds such as compounds 142Ato 152A).

For example, considering elaborated analogs of compound 70A (thepreparation of which is illustrated in Synthetic Example 2 below):

-   -   (a)        3-[[2-[[bis[bis(2-chloroethyl)amino]phosphinyl]oxy]ethyl]thio]-N-tert-butoxycarbonyl-L-alanine        may be converted to the corresponding alaninamide by reaction        with an amine (typically in excess) in an aprotic solvent (e.g.        dichloromethane+dimethylformamide) in the presence of a coupling        reagent (e.g. HBTU). That alaninamide may then be oxidized to a        compound of formula A by any of the oxidation methods described,        and then may be deprotected at the amine as described in        Synthetic Example 2. Compounds 87A to 89A are examples of        compounds that may be made this way. Alternatively, the        alaninamide may be deprotected at the amine and reacted with,        for example, an alkane- or arenesulfonyl chloride (typically in        excess) in an aprotic solvent in the presence of an organic base        to form the corresponding sulfonamide at the alanine nitrogen,        then oxidized to form a compound of formula A. Alternatively,        the alanine may first be oxidized, then deprotected, and then        elaborated at the sulfone stage. Compounds 95A to 97A are        examples of compounds that may be made this way; and/or (b) an        ester of        3-[[2-[[bis[bis(2-chloroethyl)amino]phosphinyl]oxy]ethyl]thio]-N-tert-butoxycarbonyl-L-alanine        (for example, prepared by starting the synthesis with the        corresponding L-cysteine ester) may be converted to the        sulfonamide at the alanine nitrogen, and then oxidized to form a        compound of formula A. Compounds 79A to 81A are examples of        compounds that may be made this way. The esters may be        hydrolyzed to give the corresponding acids. Compounds 103A to        105A are examples of compounds that may be made this way.        Alternatively, the alanine ester may be oxidized, then        deprotected, and then elaborated at the sulfone stage.

Compounds of Formula A.

Synthetic Example 1 Preparation ofN-methyl-2-[[2-[[bis[bis(2-chloroethyl)amino]phosphinyl]-oxy]ethyl]sulfonyl]acetamide,compound 13A

N-methyl-2-[[2-[[bis[bis(2-chloroethyl)amino]phosphinyl]oxy]ethyl]thio]acetamide.To a solution of crushed sodium hydroxide pellets, 2.28 g (57 mmol), in20 mL anhydrous methanol cooled in an ice bath was addedN-methyl-2-mercaptoacetamide, 3 g (28.5 mmol), slowly, followed by theaddition of 20 mL anhydrous toluene and2-(4-bromobenzenesulfonyloxy)ethyltetrakis(2-chloroethyl)-phosphorodiamidate, 34.4 mL of 0.83 M solutionin toluene (28.5 mmol), while maintaining the temperature at 5° C. orless. After stirring for about 30 min, the reaction mixture became awhite slurry. It was stirred at 5° C. to room temperature overnight.LC-MS analysis indicated all the starting material,N-methyl-2-mercaptoacetamide, was consumed and the desired product wasformed as a major product. The mixture was filtered and the filtrate wasconcentrated. The residue was partitioned between diisopropyl acetateand brine. The aqueous phase was further extracted with diisopropylacetate three times, and the combined organic phases were dried (MgSO₄)and evaporated to give the crude product, which was purified by columnchromatography on a silica column using hexane/acetate andacetate/methanol as eluting solvents to affordN-methyl-2-[[2-[[bis[bis(2-chloroethyl)amino]phosphinyl]oxy]ethyl]thio]acetamideas a light yellow oil, 12 g (88% yield). ¹H NMR PMSO): δ 7.90 (bs, 1H)4.05 (m, 2H), 3.71 (m, 8H), 3.33 (m, 8H), 3.12 (s, 2H), 2.87 (m, 2H),2.59 (d, 3H, J=4.7 Hz), 2.76 (m, 1H). Mass spectrum (LC-MS): m/z 496[C₁₃H₂₆Cl₄N₃O₃PS+H]⁺.

N-methyl-2-[[2-[[bis[bis(2-chloroethyl)amino]phosphinyl]oxy]ethyl]sulfonyl]acetamide.N-methyl-2-[[2-[[bis[bis(2-chloroethyl)amino]phosphinyl]oxy]ethyl]thio]acetamide,7.4 g (15.6 mmol), was dissolved in 5 mL dichloromethane in a 50-mLround-bottom flask, and the solution was cooled in ice bath. Peraceticacid, 7.4 g of 32 wt. % (31.2 mrmol) was added slowly and the reactionmixture was stirred at 0° C. to room temperature overnight. LC-MSanalysis indicated the reaction was complete with no starting materialremaining. The solvent was evaporated and the oily residue waspartitioned between isopropyl acetate and aqueous sodium bicarbonate.The organic phase was further washed with aqueous sodium bicarbonate andbrine, dried (MgSO₄) and evaporated to give the product as an oil.Treatment with ethyl ether caused the precipitation of a white solid,which was collected by filtration, washed with a small amount of ethylether and dried under high vacuum to give the final product,N-methyl-2-[[2-[[bis[bis(2-chloroethyl)amino]phosphinyl]oxy]ethyl]sulfonyl]acetamide,compound 13A, 5.37 g (68%). The filtrate was saved for laterpurification. HPLC coupled with ELSD (Evaporative Light ScatteringDetection) analysis of the product showed a purity of 99%. ¹H NMR(CDCl₃): δ 6.59 (bs, 1H), 4.53 (m, 2H), 3.95 (s, 2H), 3.65 (m, 10H),3.47 (m, 8H), 2.88 (d, 3H, J=4.7 Hz). ³¹P NMR (CDCl₃): δ 17.7 (s, 1P).Mass spectrum (LC-MS): m/z 508 [C₁₃H₂₆Cl₄N₃O₅PS+H]⁺.

Synthetic Example 2 Preparation of3-[[2-[[bis[bis(2-chloroethyl)amino]phosphinyl]oxy]ethyl]-sulfonyl]-L-alanine,compound 70A, as its hydrochloride salt

3-[[2-[[Bis[bis(2-chloroethyl)amino]phosphinyl]oxy]ethyl]thio]-N-tert-butoxycarbonyl-L-alanine.Crushed sodium hydroxide pellets, 1.2 g (29.8 mmol), and 30 mL methanolwere added to a 100-mL round-bottom flask, and the mixture stirred untilthe sodium hydroxide had dissolved. The solution was then cooled to 5°C., and N-tert-butoxycarbonyl-L-cysteine, 3 g (13.6 mmol), and2-(4-bromobenzenesulfonyloxy)ethyltetrakis(2-chloroethyl)phosphorodiamidate, 8.25 mL of 1 M solution intoluene (8.25 mmol), were added while maintaining the temperature at 5°C. or less. The reaction mixture was stirred under nitrogen for 2 hoursat room temperature, and after about 40 minutes, the solution became awhite slurry. HPLC analysis of the reaction mixture showed 96.7%3-[[2-[[bis[bis(2-chloroethyl)amino]phosphinyl]oxy]ethyl]thio]-N-tert-butoxycarbonyl-L-alanine,3.3% di-N-tert-butoxycarbonyl-L-cystine, and no remaining2-(4-bromobenzenesulfonyloxy)ethyltetrakis(2-chloroethyl)phosphorodiamidate. The pH of the mixture wasadjusted to 6-7 with 1 M phosphoric acid, and the mixture wasconcentrated under high vacuum to a white residue, 9.22 g. The residuewas dissolved in 30 mL water and washed with 30 mL and 15 mL isopropylacetate. The aqueous layer was diluted with 38 mL isopropyl acetate andthe pH of the mixture adjusted to 5.15-5.20 with 1 M phosphoric acid.The layers were separated and the isopropyl acetate layer retained. Theaqueous layer was extracted twice more with isopropyl acetate, and theisopropyl acetate extracts combined, washed with water (2×3.5 mL, 2×10.5mL), and concentrated under high vacuum to give3-[[2-[[bis[bis(2-chloroethyl)amino]phosphinyl]oxy]ethyl]thio]-N-tert-butoxycarbonyl-L-alanineas a clear viscous oil, 5.60 g (70% yield). HPLC analysis of the productshowed a purity of 97.3%. ¹H NMR: δ 7.05-7.18 (m, 1H), 4.04 (m, 3H),3.70 (m, 8H), 3.33 (m, 8H), 2.92 (m, 1H), 2.81 (m, 1H), 2.76 (m, 1H).¹³C NMR: δ 172.3, 172.0, 155.3, 78.2, 66.9, 63.7, 56.1, 53.7, 48.4,48.4, 42.3, 32.8, 31.9, 31.8, 28.1, 25.4, 21.5. ³¹P NMR: δ 17.1 (s, 1P).Mass spectrum: m/z 592 [C₁₈H₃₄Cl₄N₃O₆PS—H]⁻, 777,439.

3-[[2-[[Bis[bis(2-chloroethyl)amino]phosphinyl]oxy]ethyl]sulfonyl]-N-tert-butoxycarbonyl-L-alanine.3-[[2-[[bis[bis(2-chloroethyl)amino]phosphinyl]oxy]ethyl]thio]-N-tert-butoxycarbonyl-L-alanine,2.5 g (4.21 mmol), was dissolved in 15 mL isopropyl acetate in a 50-mLround-bottom flask, and the solution was cooled to 5° C. Peracetic acid,2.22 mL of 32 wt. % (2.5 g, 10.5 numol), was added, and the reactionmixture was stirred for 7.5 hours at 0-5° C. After 5 hours, HPLCanalysis of the reaction mixture showed no starting material. Thereaction mixture was washed with water (2×6.5 mL), aqueous sodiumhydrosulfite (6.5 mL of 1 M), and again with water (2×6.5 mL). Theisopropyl acetate layer tested negative for peroxides with starch/iodinepaper, and was concentrated under high vacuum overnight to give3-[[2-[[bis[bis(2-chloroethyl)amino]phosphinyl]oxy]ethyl]sulfonyl]-N-tert-butoxycarbonyl-L-alanineas a white, sticky foam, 1.94 g (74% yield). HPLC analysis of theproduct showed a purity of 96.7%. ¹H NMR: δ 7.29-7.38 (m, 1H), 4.43 (m,1H), 4.29 (m, 2H), 3.71 (m, 8H), 3.57 (m, 4H), 3.34 (m, 8H), 1.39 (s,9H). ¹³C NMR: δ 171.9, 171.2, 155.1, 78.6, 66.9, 58.0, 54.2, 53.6, 53.5,48.3, 48.3, 42.3, 28.1, 21.6, 21.0, 21.0. ³¹P NMR: δ 17.4 (s, 1P). Massspectrum: m/z 624 [C₁₈H₃₄Cl₄N₃O₈PS−H]⁻.

3-[[2-[[Bis[bis(2-chloroethyl)amino]phosphinyl]oxy]ethyl]sulfonyl]-L-alanine.3-[[2-[[Bis[bis(2-chloroethyl)amino]phosphinyl]oxy]ethyl]sulfonyl]-N-tert-butoxycarbonyl-L-alanine,1.82 g (2.91 mmol), was dissolved in 18 mL ethyl acetate, and to thissolution was added hydrogen chloride, 1.75 mL of 2 M solution in diethylether (3.5 mmol); and the resulting mixture stirred under nitrogenovernight at room temperature. HPLC analysis of the reaction mixtureshowed that the reaction was incomplete (20.8% product), so anadditional 0.9 equivalents of hydrogen chloride solution was added andthe reaction mixture stirred for an additional 4 days, resulting in theformation of a slurry. HPLC analysis of the reaction mixture showed99.7% product and 0.3% starting material. The slurry was filtered undernitrogen, washed with ethyl acetate, and dried under nitrogen for 3hours to give3-[[2-[[bis[bis(2-chloroethyl)amino]phosphinyl]oxy]ethyl]sulfonyl]-L-alanine,compound 70A, as the hydrochloride salt, off-white solid, 0.90 g (55%yield). HPLC analysis of the product showed a purity of 99.7%. ¹H NMR: δ8.50-9.10 (bs, 3H), 4.43 (m, 1H), 4.32 (m, 2H), 3.93-3.97 (m, 1H),3.70-3.80 (m, 11H), 3.34 (m, 8H). ¹³C NMR: δ 168.2, 58.0, 57.9, 53.8,53.7, 53.0, 48.3, 48.3, 47.1, 42.3. ³¹P NMR: δ 17.5 (s, 1P). Massspectrum: m/z 526 [C₁₃H₂₆Cl₄N₃O₆PS+H]⁺.

Synthetic Example 3 Preparation ofα-[[2-[[bis[bis(2-chloroethyl)amino]phosphinyl]oxy]ethyl]-sulfonyl]toluene,compound 128A

α-[[2-[[3is [bis(2-chlotoethyl)amino]phosphinyl]oxy]ethyl]thio]toluene.Sodium hydroxide, 6.4 g (161 mmol), was dissolved in 100 mL methanolunder nitrogen at room temperature, and α-toluenethiol, 9.52 mL (10 g,80.5 mmol), was added. After the resulting mixture was stirred for 5minutes, 2-(4-bromobenzenesulfonyloxy)ethyltetrakis(2-chloroethyl)phosphorodiamidate, 80.5 mL of 1 M solution intoluene (80.5 mmol), was added with stirring. A precipitate formedwithin 1 minute of the addition, and stirring was continued. Analysis ofthe reaction after 2 hours showed that the reaction was nearly complete,and stirring was continued overnight at room temperature. Analysis thenshowed that the reaction was complete, and stirring was stopped and thereaction mixture allowed to stand and then filtered to remove theprecipitated sodium 4-bromobenzenesulfonate. The methanol was removedfrom the filtrate by evaporation and the residue was dissolved in 250 mLisopropyl acetate. The isopropyl acetate solution was washed withaqueous sodium hydroxide (100 mL of 1 M), water (100 mL), and brine (100mL), and was then dried over anhydrous sodium sulfate and filtered. Theisopropyl acetate was removed by vacuum evaporation to give an oil,which was dried under vacuum at 40° C. overnight, giving 40.4 g ofα-[[2-[[bis[bis(2-chloroethyl)amino]phosphinyl]oxy]ethyl]thio]toluene asan orange oil containing residual toluene. HPLC analysis of the productshowed a purity of 96.8%. ¹H NMR: δ 7.16-7.33 (m, 5H), 4.04-4.06 (m,2H), 3.74 (s, 2H), 3.60-3.65 (m, 8H), 3.36-3.43 (m, 8H), 2.69 (t, 2H).

α-[[2-[[Bis[bis(2-chloroethyl)amino]phosphinyl]oxy]ethyl]sulfonyl]toluene.α-[[2-[[Bis[bis(2-chloroethyl)amino]phosphinyl]oxy]ethyl]thio]toluene,9.2 g (18.7 mmol), was dissolved in 20 mL concentrated acetic acid in a100-mL round-bottom flask, and the solution cooled to 5° C. Peraceticacid, 7.9 mL of 32 wt. % (8.9 g, 37.5 mmol), was added dropwise over 20minutes. The reaction mixture was allowed to warm to room temperatureand stirred for 16 hours, and was then concentrated under reducedpressure on a rotary evaporator to give a yellow oil. The oil wasdissolved in 100 mL dichloromethane, and 300 mL hexanes was added,giving a milky white solution, which was allowed to stand for 18 hours.The solution was vacuum filtered and the white solid residue was washedwith hexanes (3×50 mL), then dried under high vacuum for 18 hours togiveα-[[2-[[bis[bis(2-chloroethyl)amino]phosphinyl]oxy]ethyl]sulfonyl]toluene,as a white solid, 8.2 g (15.5 mmol, 83% yield). HPLC analysis of theproduct showed a purity of 96.4%. ¹H NMR: δ 7.40-7.42 (m, 5H), 4.54 (s,2H), 4.24-4.29 (q, 2H), 3.67-3.73 (m, 8H), 3.51 (t, 2H), 3.29-3.34 (m,8H). ¹³C NMR: δ 131.9, 129.2, 128.8, 59.6, 58.9, 49.0, 43.0, 40.8, 39.6.³¹P NMR: δ 17.3 (s, 1P). Mass spectrum: m/z 529 [C₁₇H₂₇Cl₄N₂O₄PS+H]⁺.

Synthetic Example 4 Preparation ofN-{2-[[2-[[bis[bis(2-chloroethyl)amino]phosphinyl]oxy]-ethyl]sulfonyl]acetyl}-L-phenylalanine,compound 15A

2-[[2-[[bis[bis(2-chloroethyl)amino]phosphinyl]oxy]ethyl]thio]aceticacid. Methyl 2-mercapto-acetate, 2.6 mL (28.5 mmol), was dissolved in 10mL dry methanol, and sodium hydroxide, 2.24 mL of 25 M solution (56mmol), and 10 mL methanol were added, followed by2-(4-bromobenzene-sulfonyloxy)ethyltetrakis(2-chloroethyl)phosphorodiamidate, 26 mL of 1 M solution intoluene (26 mmol) with stirring, and a further 20 mL methanol. A whiteprecipitate formed immediately, and a further 30 mL methanol was addedto enable easy stirring overnight. The solid was filtered, and anadditional 2 mL of the sodium hydroxide solution added to the filtrateand stirred for 2 hours. The basic solution was washed with ethylacetate, then hydrochloric acid added until the solution became cloudy.This solution was extracted three times with ethyl acetate, and thecombined ethyl acetate extracts backwashed with water, dilutehydrochloric acid, water, and brine, and then dried over Na₂SO₄ andconcentrated, to give 7.31 g of2-[[2-[[bis[bis(2-chloroethyl)amino]phosphinyl]oxy]ethyl]thio]aceticacid as an oil that solidified on standing. The identity was verified by¹H NMR.

N-{2-[[2-[[bis[bis(2-chloroethyl)amino]phosphinyl]oxy]ethyl]thio]acetyl}-L-phenylalanine.L-Phenylalanine tert-butyl ester hydrochloride, 1.2 mg (4.5 mmol), waspartitioned between ethyl acetate and aqueous sodium bicarbonate, washedwith water and brine, then dried. This was added to2-[[2-[[bis[bis(2-chloroethyl)amino]phosphinyl]oxy]ethyl]thio]aceticacid, 1.76 g (3.8 mmol), followed by 1.4 mL diisopropylethylamine (8.0mmol) and HBTU, 1.46 g (3.85 mmol). The mixture was stirred for severalhours at room temperature. The mixture was filtered, washed with 1 Msodium hydroxide, potassium bisulfate, and brine; then treated wit 25%trifluoroacetic acid in dichloromethane to hydrolyze the tert-butylester. The mixture was then concentrated to giveN-{2-[[2-[[bis[bis(2-chloroethyl)amino]-phosphinyl]oxy]ethyl]thio]acetyl}-L-phenylalanine.

N-{2-[[2-[[bis[bis(2-chloroethyl)amino]phosphinyl]oxy]ethyl]sulfonyl]acetyl}-L-phenylalanine.TheN-{2-[[2-[[bis[bis(2-chloroethyl)amino]phosphinyl]oxy]ethyl]thio]acetyl}-L-phenylalanineprepared above was dissolved in 20 mL ethyl acetate and oxidized using 3mL 32% peracetic acid using the procedure of Synthetic Example 1. Afterwashing the ethyl acetate solution with sodium hydrosulfite, water, andbrine, it was dried, concentrated, chromatographed, and the fractionscombined, stirred with ethanol, and lyophilized to giveN-{2-[[2-[[bis[bis(2-chloroethyl)amino]phosphinyl]oxy]ethyl]sulfonyl]-acetyl}-L-phenylalanine,1.5 g (62% yield), as a white solid. Purity was verified.

Synthetic Example 5 Preparation ofα-[[2-[[bis[bis(2-chloroethyl)amino]phosphinyl]oxy]ethyl]-sulfonyl]-4-[(ethoxycarbonylmethyl)aminocarbonyl]toluene,compound 172A

α-[[2-[[Bis[bis(2-chloroethyl)amino]phosphinyl]oxy]ethyl]thio]-4-carboxytoluene.4-(Thiomethyl)benzoic acid was prepared from methyl4-(bromomethyl)benzoate using the procedure of Org. Syn. Coll. Vol. 3,page 363 (reaction with thiourea in ethanol, followed by hydrolysis ofthe ester with aqueous sodium hydroxide). 4-(Thiomethyl)benzoic acid, 2g, was alkylated with 2-(4-bromobenzenesulfonyloxy)ethyltetrakis(2-chloroethyl)phosphorodiamidate, 15.8 mL of 0.83 M, in 10 mLmethanol and 6.25 mL of 4 M sodium hydroxide in methanol. The resultingproduct after filtration and concentration was a pale yellow oil, whichwas dissolved in saturated aqueous sodium bicarbonate and washed twicewith 100 mL isopropyl acetate. The aqueous extracts were combined,acidified to pH ˜1 with sulfuric acid, and back extracted twice withinto 100 mL isopropyl acetate. The isopropyl acetate layers werecombined, dried over MgSO₄, filtered, and concentrated under vacuum togiveα-[[2-[[bis[bis(2-chloroethyl)amino]phosphinyl]oxy]ethyl]thio]-4-carboxytolueneas a pale yellow oil, 5.4 g, with product identity verified by LC-MS.

α-[[2-[Bis[bis(2-chloroethyl)amino]phosphinyl]oxy]ethyl]thio]-4-[(ethoxycarbonylmethyl)-aminocarbonyl]toluene.Toα-[[2-[[Bis[bis(2-chloroethyl)amino]phosphinyl]oxy]ethyl]thio]-4-carboxytoluene,411 mg, in 10 mL dichloromethane was added 400 IL diisopropylethylamineand glycine ethyl ester, 212 mg. The solution was cooled to 0° C. andHBTU, 317 mg, in 2 mL dimethylformamide was added, after which themixture was allowed to warm to room temperature. The reaction mixturewas diluted with dichloromethane, then washed 3 times with 1 Mhydrochloric acid, 5 times with saturated aqueous sodium bicarbonate,and once with brine. The organic layer was dried over MgSO₄, filtered,and the solvents removed under vacuum to giveα-[[2-[[bis[bis(2-chloroethyl)amino]phosphinyl]oxy]ethyl]thio]-4-[(ethoxycarbonylmethyl)aminocarbonyl]tolueneas a clear oil, 411 mg, with product identity verified by LC-MS.

α-[[2-[[bis[bis(2-chloroethyl)amino]phosphinyl]oxy]ethyl]sulfonyl]-4-[(ethoxycarbonylmethyl)-aminocarbonyl]toluene.Theα-[[2-[[bis[bis(2-chloroethyl)amino]phosphinyl]oxy]ethyl]thio]-4-[(ethoxycarbonylmethyl)aminocarbonyl]toluene,411 mg, was dissolved in 6 mL isopropyl acetate and oxidized with 414 μL32% peracetic acid solution as described in previous Synthetic Examples.After workup,α-[[2-[[bis[bis(2-chloroethyl)amino]phosphinyl]oxy]ethyl]sulfonyl]-4-[(ethoxycarbonylmethyl)-aminocarbonyl]toluenewas isolated as a clear oil, 449 mg. Product identity from a parallelrun was verified by LC-MS.

Synthetic Example 6 Preparation ofN-(methanesulfonyl)-3-[[2-[[bis[bis(2-chloroethyl)amino]-phosphinyl]oxy]ethyl]sulfonyl]-L-alaninemethyl ester, compound 79A

3-[[2-[[Bis[bis(2-chloroethyl)amino]phosphinyl]oxy]ethyl]thio]-L-alaninemethyl ester.3-[[2-[[Bis[bis(2-chloroethyl)amino]phosphinyl]oxy]ethyl]thio]-N-tert-butoxycarbonyl-L-alanine,5.0 g (8.4 mmol), prepared as in Synthetic Example 2, was dissolved in35 mL dichloromethane, and 5.3 mL (42 mmol) chlorotrimethyl silane addedwith stirring at 0° C., followed by 80 mL methanol. With continuedstirring, the mixture was allowed to warm to room temperature, thenstirred overnight. The solvents were then removed under vacuum to give alight yellow oil. This was dissolved in ethyl acetate and precipitatedwith diethyl ether. Filtration and concentration of the filtrate gavetwo fractions of3-[[2-[[bis[bis(2-chloroethyl)amino]phosphinyl]oxy]ethyl]thio]-L-alaninemethyl ester, totaling 4.87 g. Purity (96%) and identity were verifiedby ELSD and LC-MS (m/z 508 (M+2+H)⁺).

N-(Methanesulfonyl)-3-[[2-[[bis[bis(2-chloroethyl)amino]phosphinyl]oxy]ethyl]thio]-L-alaninemethyl ester.3-[[2-[[Bis[bis(2-chloroethyl)amino]phosphinyl]oxy]ethyl]thio]-L-alaninemethyl ester, 400 mg (0.8 mmol), was dissolved in 4 mL anhydroustetrahydrofuran, and 550 μL (3.2 mmol) diisopropylethylamine added,followed by methanesulfonyl chloride, 181 mg (1.6 mmol). The reactionmixture was purged with argon and stirred overnight. After adding afurther 275 μL (1.6 mmol) diisopropylethylamine, thentris(2-aminoethyl)amine-polystyrene resin, 480 mg (1.6 mmol), themixture was agitated for 6 hours, then filtered and the filtrateevaporated to dryness under vacuum, givingN-(methanesulfonyl)-3-[[2-[[bis[bis(2-chloroethyl)amino]phosphinyl]oxy]ethyl]thio]-L-alaninemethyl ester, 397 mg. Purity was verified with ELSD and identityverified with ¹H and ³¹P NMR.

N-(Methanesulfonyl)-3-[[2-[[bis[bis(2-chloroethyl)amino]phosphinyl]oxy]ethyl]sulfonyl]-L-alaninemethyl ester.N-(Methanesulfonyl)-3-[[2-[[bis[bis(2-chloroethyl)amino]phosphinyl]oxy]ethyl]thio]-L-alaninemethyl ester, 369 mg (0.63 mmol), was dissolved in 5 mL ethyl acetateand oxidized with 662 μL 32% peracetic acid solution as described inprevious Synthetic Examples. After workup,N-(Methanesulfonyl)-3-[[2-[[bis[bis(2-chloroethyl)amino]phosphinyl]oxy]ethyl]sulfonyl]-L-alaninemethyl ester, compound 79A, was isolated as a clear oil, 358 mg (870%).Purity was verified with ELSD and identity verified with LC-MS (m/z 618(N+2+H)⁺).

Other compounds of formula A as shown in the tables below were preparedby similar methods. Note that the largest peak in the mass spectrum forcompounds of formula A is at 2 greater than the peak corresponding tothe exact mass (the mass determined based on the most common isotopes ofthe atoms making up the molecule) because of the presence of 4 chlorineatoms in the molecule: the intensities of the three largest peaks are3:4:2 at M, M+2, and M+4.

The following compounds of formula A were prepared: Compound StructureExact mass MS  1A

464  2A

462 463 (M + H⁺)  3A

478 479 (M + H⁺)  4A

478  5A

492 495 (M+ 2 + H⁺)  6A

492  7A

492 493 (M + H⁺)  8A

506  9A

506 10A

504 11A

518 12A

548 549 (M + H⁺) 13A

507 508 (M + H⁺) 14A

494 15A

641 642 (M + H⁺) 16A

480 481 (M + H⁺) 17A

521 524 (M + 2 + H⁺) 18A

728 729 (M + H⁺) 19A

652 653 (M + H⁺) 20A

615 615 (M + H⁺) 21A

682 683 (M + H⁺) 22A

576 577 (M + H⁺) 23A

606 607 (M + H⁺) 24A

570 571 (M + H⁺) 25A

697 698 (M + H⁺) 26A

641 644 (M + 2 + H⁺) 27A

780 680 (M − BOC) 28A

623 624 (M + H⁺) 29A

508 509 (M + H⁺) 30A

627 628 (M + H⁺) 31A

622 32A

608 33A

613 34A

565 568 (M + 2 + H⁺) 35A

683 630 (M + 2 − Bu + H⁺) 36A

627 630 (M + 2 + H⁺) 37A

621 622 (M + 2 − H⁻) 38A

607 608 (M + 2 − H⁻) 39A

565 568 (M + H) 40A

551 554 (M + H) 41A

664 665 (M + 2 − H⁻) 42A

647 648 (M + 2 − H⁻) 43A

649 650 (M + 2 − H⁻) 44A

621 622 (M + 2 − H⁻) 45A

664 665 (M + 2 − H⁻) 46A

713 714 (M + 2 −H⁻⁾ 47A

676 677 (M + 2 − H⁻⁾ 48A

551 552 (M + H⁺) 49A

537 538 (M + H⁺) 50A

521 522 (M + H⁺) 51A

563 564 (M + H⁺) 52A

577 578 (M + H⁺) 53A

521 522 (M + H⁺) 54A

535 536 (M + H⁺) 55A

547 548 (M + H⁺) 56A

551 552 (M + H⁺) 57A

565 566 (M + H⁺) 58A

577 578 (M + H⁺) 59A

591 592 (M + H⁺) 60A

533 534 (M + H⁺) 61A

608 611 (M + 2 + H⁺) 62A

591 594 (M + 2 + H⁺) 63A

593 596 (M + 2 + H⁺) 64A

565 568 (M + 2 + H⁺) 65A

608 611 (M + 2 + H⁺) 66A

657 660 (M + 2 + H⁺) 67A

633 636 (M + 2 + H⁺) 68A

562 565 (M + 2 + H⁺) 69A

606 607 (M + H⁺) 70A

523 526 (M + 2 + H⁺) 71A

623 72A

551 73A

565 568 (M + 2 + H⁺) 74A

593 594 (M + H⁺) 75A

551 552 (M + H⁺) 76A

551 552 (M + H⁺) 77A

537 78A

522 523 (M + H⁺) 79A

615 618 (M + 2 + H⁺) 80A

677 680 (M + 2 + H⁺) 81A

711 714 (M + 2 + H⁺) 82A

612 615 (M + 2 + H⁻) 83A

692 595 (M + 2 − BOC + H⁺) 84A

664 665 (M + H⁺) 85A

785 787 (M + 2⁺) 86A

666 668 (M + 2⁺) 87A

592 594 (M + 2⁺) 88A

566 568 (M + 2⁺) 89A

564 566 (M + 2⁺) 90A

685 687 (M + 2⁺) 91A

752 753 (M + H⁺) 92A

704 706 (M + 2⁺) 93A

825 826 (M + H⁺) 94A

594 597 (M + 2 + H⁺) 95A

690 691 (M + H⁺) 96A

670 671 (M + H⁺) 97A

642 643 (M + H⁺) 98A

763 765 (M + 2⁺) 99A

777 778 (M + H⁺) 100A

757 759 (M + 2⁺) 101A

729 730 (M + H⁺) 102A

850 852 (M + 2⁺) 103A

663 664 (M + H⁺) 104A

601 602 (M + 2 − H⁻) 105A

697 698 (M + 2 − H) 106A

546 549 (M + 2 + H⁺) 107A

526 527 (M + H⁺) 108A

546 549 (M + 2 + H⁺) 109A

580 581 (M + H⁺) 110A

526 527 (M + H⁺) 111A

530 531 (M + H⁺) 112A

580 583 (M + 2 + H⁺) 113A

588 589 (M + H⁺) 114A

568 571 (M + 2 + H⁺) 115A

542 545 (M + 2 + H⁺) 116A

580 583 (M + 2 + H⁺) 117A

512 515 (M + 2 + H⁺) 118A

556 557 (M + H⁺) 119A

556 555 (M − H⁻) 120A

513 514 (M + H⁺) 121A

569 570 (M + H⁺) 122A

516 517 (M + H⁺) 123A

528 529 (M + H⁺) 124A

513 514 (M + H⁺) 125A

554 555 (M + H⁺) 126A

557 560 (M + 2 + H⁺) 127A

554 555 (M + H⁺) 128A

526 528 (M + 2⁺) 129A

540 543 (M + 2 + H⁺) 130A

540 543 (M + 2 + H⁺) 131A

560 563 (M + 2 + H⁺) 132A

544 547 (M + 2 + H⁺) 133A

594 597 (M + 2 + H⁺) 134A

595 598 (M + 2 + H⁻) 135A

595 598 (M + 2 + H⁺) 136A

560 563 (M + 2 + H⁺) 137A

582 585 (M + 2 + H⁺) 138A

571 574 (M + 2 + H⁺) 139A

570 569 (M − H⁻) 140A

584 585 (M + H⁺) 141A

541 544 (M + 2 + H⁺) 142A

641 644 (M + 2 + H⁺) 143A

598 601 (M + 2 + H⁺) 144A

698 601 (M + 2 − BOC + H⁺) 145A

826 146A

752 655 (M + 2 − BOC + H⁺) 147A

670 673 (M + 2 + H⁺) 148A

652 655 (M + 2 + H⁺) 149A

652 653 (M + H⁺) 150A

668 669 (M + H⁺) 151A

682 683 (M + H⁺) 152A

698 699 (M + H⁺) 153A

551 552 (M + H⁺) 154A

639 155A

597 598 (M + H⁺) 156A

623 624 (M + H⁺) 157A

625 626 (M + H⁺) 158A

627 628 (M + H⁺) 159A

637 638 (M + H⁺) 160A

613 614 (M + H⁺) 161A

659 660 (M + H⁺) 162A

569 570 (M + H⁺) 163A

597 598 (M + H⁺) 164A

608 609 (M + H⁺) 165A

639 640 (M + H⁺) 166A

653 654 (M + H⁺) 167A

680 681 (M + H⁺) 168A

583 584 (M + H⁺) 169A

657 658 (M + H⁺) 170A

680 681 (M + H⁺) 171A

732 733 (M + H⁺) 172A

655 656 (M + H⁺) 173A

640 641 (M + H⁺) 174A

660 661 (M + H⁺) 175A

696 697 (M + H⁺) 176A

715 716 (M + H⁺) 177A

728 729 (M + H⁺) 178A

655 656 (M + H⁺) 179A

516 517 (M + H⁺) 180A

532 533 (M + H⁺) 181A

527 528 (M + H⁺) 182A

543 544 (M + H⁺) 183A

587 588 (M + H⁺) 184A

603 604 (M + H⁺) # represents thebis[bis(2-chloroethyl)amino]phosphinyl]oxy]ethyl]sulfonyl group of theformula

attached to the rest of the molecule at the sulfur atom. t-BOCrepresents the tert-butoxycarbonyl group.

All of these compounds of formula A were analyzed to confirm identityand purity, using HPLC for purity, and one or more of mass spectrometryand NMR (¹H, ¹³C, and/or ³¹P) for identity, and were confirmed to be theexpected product in good purity. Other compounds of formula A may besimilarly prepared.

Compounds of Formula BB and Formula B.

Synthetic Example 7 Preparation of1,5-di[[2-[[bis[bis(2-chloroethyl)amino]phosphinyl]oxy]-ethyl]sulfonyl]naphthalene(compound 23B)

1,5-Di[[2-[[bis[bis(2-chloroethyl)amino]phosphinyl]oxy]ethyl]thio]naphthalene.1,5-Naphthalenedithiol, 194 mg (1.009 mmol), was dissolved in 10 mLmethanol and sodium hydroxide, 2 mL of 2 M solution in methanol (4mmol); and 2-(4-bromobenzenesulfonyloxy)ethyltetrakis(2-chloroethyl)phosphorodiamidate, 1.86 mL of 1 M solution intoluene (1.86 mmol) was added. The reaction mixture was stirred at roomtemperature, and after about 30 minutes a white precipitate had formed.The mixture was stirred overnight, then the precipitate filtered and thefiltrate concentrated to a solid residue, which was extracted into ethylacetate and washed with 1 N aqueous sodium hydroxide, water, and brine,then dried over anhydrous magnesium sulfate. The ethyl acetate wasremoved under vacuum, and the crude1,5-di[[2-[[bis[bis(2-chloroethyl)amino]phosphinyl]oxy]ethyl]-thio]naphthalenewas purified by flash chromatography (silica column, eluting with agradient between 75% ethyl acetate/hexane and 100% ethyl acetate). Thefinal1,5-di[[2-[[bis[bis(2-chloroethyl)amino]-phosphinyl]oxy]ethyl]thio]naphthalene(compound 23BB) was a light yellow oil, yield 405 mg (42%), with productidentity and purity verified by HPLC and NMR. Mass spectrum: m/z 937[C₃₀H₄₆Cl₈N₄O₄P₂S₂+H⁺].

1,5-Di[[2-[[bis[bis(2-chloroethyl)amino]phosphinyl]oxy]ethyl]sulfonyl]naphthalene.1,5-di[[2-[[bis[bis(2-chloroethyl)amino]phosphinyl]oxy]ethyl]thio]naphthalene,324 mg (0.346 mmol) was dissolved in 10 mL ethyl acetate and cooled to0-5° C. in an ice bath. To this was added 365 μL peracetic acid (32% byweight, 1.735 mmol), and the mixture kept at 0-5° C. for 1 hour, thenallowed to warm to room temperature and allowed to stand overnight. Thereaction mixture was dissolved in ethyl acetate, and the ethyl acetatelayer separated, washed twice with water, twice with aqueous 1 M sodiumdithionite, and twice more with water. The ethyl acetate was removedunder vacuum, and the residue suspended in diethyl ether and sonicatedfor 30 minutes, then the ether poured off, with this procedure repeatedonce more. The1,5-di[[2-[[bis[bis(2-chloroethyl)amino]phosphinyl]oxy]ethyl]sulfonyl]-naphthalene,compound 23B, was isolated as a white solid, 260 mg, with productidentity and purity verified by HPLC and NMR. Mass spectrum: m/z 1001[C₃₀H₄₆Cl₈N₄O₈P₂S₂+4+H³⁰ ].

Other compounds of formula BB and formula B as shown in the tables belowwere prepared by similar methods. Note that the largest peak in the massspectrum for compounds of formula B and formula BB (and also forcompounds of formula C and formula CC) is at 4 greater than the peakcorresponding to the exact mass because of the presence of 8 chlorineatoms in the molecule: the intensities of the 4 largest peaks are4:9:10:6 at M, M+2, M+4, and M+6.

The following compounds of formula BB were prepared: Compound Formula 1BB

 2BB

 3BB

 4BB

 5BB

 6BB

 7BB

 8BB

 9BB

10BB

11BB

12BB

13BB

14BB

15BB

16BB

17BB

18BB

19BB

20BB

21BB

22BB

23BB

$ represents the bis[bis (2-chloroethyl)amino]phosphinyl]oxy]ethyl]thiogroup of the formula

attached to the rest of the molecule at the sulfur atom

All of these compounds of formula BB were analyzed to confirm identityand purity, using HPLC for purity, and one or more of mass spectrometryand NMR (¹H, ¹³C, and/or ³¹P) for identity, and were confirmed to be theexpected product in good purity.

The following compounds of formula B were prepared: Compound FormulaExact mass MS  1B

898  2B

926  929  3B

912  4B

926  6B

940  942  7B

982  985  8B

942  947 (M + 4 + H⁺)  9B

986  991 (M + 4 + H⁺) 10B

946  951 (M + 4 + H⁺) 11B

946  951 (M + 4 + H⁺) 12B

946  951 (M + 4 + H⁺) 13B

974  979 (M + 4 + H⁺) 14B

974  979 (M + 4 + H⁺) 15B

974  979 (M + 4 + H⁺) 16B

1019 1024 (M + 4 + H⁺) 17B

989  994 (M + 4 + H⁺) 18B

1089 1092 19B

1046 1051 (M + 4 + H⁺) 20B

1074 1079 (M + 4 + H⁺) 21B

1100 1105 (M + 4 + H⁺) 22B

1094 1099 (M + 4 + H⁺) 23B

996 1001 (M + 4 + H⁺) # represents thebis[bis(2-chloroethyl)amino]phosphinyl]oxy]ethyl]sulfonyl group of theformula

attached to the rest of the molecule at the sulfur atom

All of these compounds of formula B were analyzed to confirm identityand purity, using HPLC for purity, and one or more of mass spectrometryand NMR (¹H, ¹³C, and/or ³¹P) for identity, and were confirmed to be theexpected product in good purity.

Other compounds of formula BB and formula B may be similarly prepared.

Compounds of Formula CC and Formula C.

Synthetic Example 8 Preparation ofdi[2-[[bis[bis(2-chloroethyl)amino]phosphinyl]oxy]ethyl sulfone,compound 1C

N,N,N′,N′-Tetrakis(2-chloroethyl)phosphorodiamidoyl chloride.Bis(2-chloroethyl)amine hydrochloride, 7.68 g (86 mmol), and phosphoryltrichloride, 4 mL (43 mmol), were suspended in 150 mL toluene, afterwhich triethylamine, 12.6 mL (90 mmol), was added over 5 minutes. Thereaction mixture was stirred at room temperature for 18 hours undernitrogen, then additional aliquots of bis(2-chloroethyl)aminehydrochloride, 7.68 g, and triethylamine, 12.6 mL, were added. Theresulting mixture was heated to reflux and held at that temperature for8 hours. After cooling, the precipitate was filtered and the filtrateconcentrated under vacuum to remove volatiles. CrudeN,N,N′,N′-tetrakis(2-chloroethyl)phosphorodiamidoyl chloride wasobtained was obtained as a dark brown oil, 15.45 g. The crude materialwas purified by flash column chromatography using 5-10% ethylacetate/dichloromethane as eluent, and the pure fractions collected andvolatiles removed, giving a total of 8.33 gN,N,N′,N′-tetrakis(2-chloroethyl)phosphorodiamidoyl chloride as a lightyellow oil, with identity verified by 1H and ³¹P NMR.

5-Hydroxy-3-thiapentylN,N,N′,N′-tetrakis(2-chloroethyl)phosphorodiamidate.N,N,N′,N′-Tetrakis(2-chloroethyl)phosphorodiamidoyl chloride, 636 mg(1.745 mmol), and bis(2-hydroxyethyl)sulfide, 1 mL (9.67 mmol), weredissolved in 8 mL anhydrous tetrahydrofuran and cooled to 0-5° C. in anice-water bath. Potassium tert-butoxide, 1.8 mL of 1 M solution intetrahydrofuran (1.8 mmol), was added over a 10 minute period, and thereaction mixture kept at 0-5° C. for another 30 minutes. The reactionmixture was allowed to warm to room temperature and stirred at roomtemperature for 18 hours. It was then diluted with ethyl acetate; andthe ethyl acetate layer separated, washed with 1 M hydrochloric acid andbrine, then dried over anhydrous magnesium sulfate, and the ethylacetate removed under vacuum. The crude 5-hydroxy-3-thiapentylN,N,N′,N′-tetrakis(2-chloroethyl)phosphorodiamidate was purified bycolumn chromatography (silica gel, eluting with 25% ethyl acetate/hexaneand 100% ethyl acetate), and obtained as a colorless oil, 335 mg (42.6%yield), identified by HPLC and ¹H and ³¹P NMR.

Di[2-[[bis[bis(2-chloroethyl)amino]phosphinyl]oxy]ethyl]sulfide.5-Hydroxy-3-thiapentylN,N,N′,N′-tetrakis(2-chloroethyl)phosphorodiamidate, 174 mg (0.387mmol), and N,N,N′N′-tetrakis(2-chloroethyl)phosphorodiamidoyl chloride,363 mg (0.996 mmol), were dissolved in 15 mL anhydrous tetrahydrofuranand cooled to 0-5° C. in an ice-water bath. Potassium tert-butoxide, 0.4mL of 1 M solution in tetrahydrofuran (0.4 mmol), was added over a 10-15minute period, and the reaction mixture kept at 0-5° C. for another 30minutes under nitrogen. The reaction mixture was allowed to warm to roomtemperature and stirred at room temperature for 43 hours. Additionalpotassium tert-butoxide andN,N,N′,N′-tetrakis(2-chloroethyl)phosphorodiamidoyl chloride were addedover the next 4 days to drive the reaction to completion. After 4.5days, the reaction mixture was diluted with ethyl acetate; and the ethylacetate layer separated, washed with water and brine, then dried overanhydrous magnesium sulfate, and the ethyl acetate removed under vacuum.The crudedi[2-[[bis[bis(2-chloroethyl)amino]phosphinyl]oxy]ethyl]sulfide, 630 mg,was twice purified by preparative HPLC, and the product fractionscombined and lyophilized, givingdi[2-[[bis[bis(2-chloroethyl)amino]phosphinyl]oxy]ethyl]sulfide,compound 1CC, as a colorless oil, compound 1CC, 56 mg (18% yield),identified by HPLC and ¹H and ³¹P NMR. Mass spectrum: 779(C₂₀H₄₀Cl₈N₄O₄P₂S+4+H⁺).

Di[2-[[bis[bis(2-chloroethyl)amino]phosphinyl]oxy]ethyl]sulfone. Thedi[2-[[bis[bis(2-chloroethyl)amino]phosphinyl]oxy]ethyl]sulfide, 56 mg,from the previous reaction was dissolved in 10 mL ethyl acetate andcooled to 0-5° C. in an ice-water bath. Peracetic acid, 45 mL (0.214mmol), was added, and the reaction mixture held at 0-5° C. for 1 hourthen allowed to warm to room temperature and held for 2 hours tocomplete the reaction. The reaction mixture was diluted with ethylacetate, and the ethyl acetate layer separated and washed twice withwater, once with 1 M aqueous sodium dithionite, and twice more withwater. The ethyl acetate was removed under vacuum to givedi[2-[[bis[bis(2-chloroethyl)amino]phosphinyl]oxy]ethyl]sulfone,compound 1C, as a colorless oil, 54 mg (92.6% yield), identified by HPLCand ¹H and ³¹P NMR. Mass spectrum: 811 (C₂₀H₄₀Cl₈N₄O₆P₂S+4+H⁺).

In vitro Examples

The following examples illustrate the beneficial effect of thesulfonylethyl phosphorodiamidate esters of this invention against humancancer cell lines in vitro. These results are considered predictive ofefficacy in human cancer chemotherapy, as other anticancer agents testedin these assays have shown anticancer activity in humans.

The human cancer cell lines DLD-1 (colorectal adenocarcinoma), LNCap(prostate carcinoma), and OVCAR-3 (ovarian carcinoma) were obtained fromthe American Type Culture Collection, Manassas, Va., U.S.A., and HL-60(promyeloid myelocytic leukemia), MX-1 (breast carcinoma), P388, andP388ADR from the National Cancer Institute, Bethesda, Md., U.S.A.Carboplatin, doxorubicin, and melphalan were obtained from Sigma-AldrichChemical Company, St. Louis, Mo., U.S.A. The CellTiter-Glo assay kit wasobtained from Promega Corporation, Madison, Wis., U.S.A., and theCellular DNA Cytometric Analysis Reagent Kit from Roche DiagnosticsCorporation, Indianapolis, Ind., U.S.A. Lambda DNA was obtained from NewEngland Biolabs, Beverly, Mass., U.S.A., and SYBR-Gold from MolecularProbes, Inc., Eugene, Oreg., U.S.A. The DNeasy Tissue Kit was obtainedfrom Qiagen Inc., Valencia, Calif., U.S.A., and AmpliTaq Gold DNApolymerase and PCR reagents from Applied Biosystems, Foster City,Calif., U.S.A. All products were used in accordance with manufacturer'sdirections. All assays were conducted in triplicate wells, with dimethylsulfoxide (DMSO) solvent control. The extent of cell growth wasexpressed as a percentage of the signal from the solvent control wells.

In vitro Example 1 Cytotoxicity/Growth Inhibition Assay

Log-phase cells were trypsinized, collected by centrifugation, andresuspended in a small volume of fresh medium, and the density of viablecells was determined following Trypan Blue staining. Cells were dilutedin fresh media (3×10³ cells/mL for MX-1, 3×10³ cells/mL for DLD-1, and6×10³ cells/mL for LNCap cells), the test compounds (concentrationsbetween 0.1 and 200 μM, dissolved in DMSO, 50 μL) added immediatelyafter dilution to achieve a final DMSO concentration of 0.5%, then thesuspensions added at 150 μL/well to 96-well plates, and incubated forseveral hours to allow attachment in the case of adherent cells. Thecells were cultured for approximately three doubling times (3 days forMX-1 and 4 days for DLD-1 and LNCap). The cells were then collected bycentrifugation, and 100 μL of the culture supernatant was replaced bythe CellTiter-Glo reagent. After incubation for 15 minutes at roomtemperature, and the plate was read with a luminometer. A number ofcompounds of formula BB and compound 1CC were tested in this assay andfound to be active. Many compounds were found to be as potent as or morepotent than canfosfamide.

Compounds of formula A showed the following activity in this assay: IC₅₀(MX-1), IC₅₀ (DLD-1), IC₅₀ (LNCap), Compound μM μM μM  1A 16.9 9.1 57.1 2A 3.2 1.4 105.3  3A 11.4 9.2 67.6  4A 24.0 12.7 67.8  5A 14.3 6.6 42.8 6A 4.6 6.8 17.7  7A 20.6 21.8 164.7  8A 17.5 7.0 40.9  9A 5.8 5.9 17.310A 14.4 7.7 43.0 11A 10.4 4.0 18.1 12A 13.9 3.5 14.1 13A 11.7 7.1 53.514A 62.2 17.6 200.0 15A 23.6 13.7 200.0 16A 13.0 9.6 36.4 17A 6.3 4.213.9 18A 7.6 10.6 23.8 19A 14.8 8.1 17.8 20A 4.3 2.8 3.6 21A 4.2 2.8 6.322A 23.7 8.4 38.5 23A 25.9 6.6 47.5 24A 11.2 5.5 12.1 25A 5.6 3.7 18.926A 29.4 8.4 76.8 27A 6.2 3.6 15.5 28A 9.0 5.1 35.2 29A 23.0 11.5 66.430A 9.1 6.5 13.2 31A 83.7 11.3 32A 41.2 11.3 177.0 33A 20.6 8.6 98.6 34A9.0 12.0 97.2 35A 6.6 5.0 24.8 36A 12.3 5.4 200.0 37A 8.0 8.0 28.9 38A8.0 7.6 23.3 39A 30.8 13.3 40A 32.0 13.0 41A 29.3 10.8 42A 7.6 4.7 43A7.2 3.9 44A 34.8 11.1 45A 7.3 8.4 46A 21.4 8.8 47A 15.9 9.3 48A 4.4 15.549A 29.0 11.6 50A 17.1 11.9 51A 6.9 6.7 52A 15.6 10.2 53A 11.3 8.4 54A9.3 7.4 55A 14.7 10.1 56A 19.3 8.2 57A 18.0 10.1 58A 17.0 12.9 59A 25.415.1 60A 11.3 7.4 61A 30.9 20.0 62A 15.0 16.5 63A 10.0 17.2 64A 37.322.0 65A 31.8 21.4 66A 29.1 18.8 67A 2.8 1.9 68A 36.7 18.0 69A 34.6 14.170A 36.7 10.7 73A 29.6 18.0 73.2 74A 46.7 27.2 200.0 75A 33.1 7.6 184.776A 23.2 6.6 99.7 77A 21.3 8.0 139.7 78A 18.8 6.3 200.0 79A 88.0 26.680A 27.1 10.7 80.2 81A 33.3 6.9 86.6 82A 27.9 7.7 59.8 83A 12.9 6.7 27.884A 10.6 6.0 29.6 85A 11.8 8.8 41.1 86A 25.1 10.3 73.5 87A 19.5 9.7136.3 88A 17.9 9.5 125.8 89A 22.2 8.5 140.5 90A 23.8 10.4 200.0 91A 10.74.8 33.3 92A 14.7 7.3 36.1 93A 19.3 13.2 59.1 94A 22.8 37.5 200.0 95A21.3 9.9 46.6 96A 30.2 9.6 81.0 97A 26.8 8.5 57.8 98A 27.9 9.9 144.5 99A16.2 6.4 33.5 100A  16.2 7.4 61.4 101A  14.8 7.2 57.0 102A  19.9 9.281.6 103A  18.2 14.5 150.8 104A  25.3 20.0 200.0 105A  31.6 23.1 200.0106A  4.2 3.7 18.3 107A  2.5 1.6 18.1 108A  3.3 2.3 6.4 109A  1.1 0.67.7 110A  2.7 1.3 20.5 111A  1.3 0.6 20.1 112A  1.0 0.8 6.3 113A  10.92.2 18.4 114A  4.7 1.9 8.2 115A  5.3 4.1 18.5 116A  3.1 2.2 6.6 117A 4.1 4.0 16.3 118A  37.2 10.9 200.0 119A  32.5 9.8 189.0 120A  7.1 3.612.2 121A  2.6 1.2 8.5 122A  4.3 3.1 23.0 123A  9.0 5.9 25.1 124A  7.26.0 15.5 125A  22.5 7.8 45.1 126A  20.3 6.7 125.8 127A  15.2 5.8 200.0128A  4.5 3.2 129A  7.1 5.4 16.3 130A  6.5 5.1 22.0 131A  5.2 3.6 6.9132A  6.1 5.0 16.3 133A  2.7 2.6 8.2 134A  1.5 1.2 4.8 135A  3.5 0.619.1 136A  2.8 1.7 21.4 137A  1.7 0.8 22.3 138A  16.8 1.8 12.0 139A 25.3 9.3 82.1 140A  11.9 5.9 23.3 141A  17.3 12.9 45.9 142A  22.5 18.1113.5 143A  15.9 10.9 110.2 144A  5.7 4.0 17.9 145A  7.4 6.9 15.5 146A 5.1 5.6 14.2 147A  12.2 11.1 63.9 148A  200.0 200.0 149A  94.3 7.5 200.0150A  31.3 7.6 118.2 151A  28.6 6.1 158.1 152A  37.6 9.3 108.0 153A  3.01.8 6.9 154A  5.5 5.8 26.2 155A  2.6 10.7 42.4 156A  4.8 3.5 31.4 157A 3.1 2.3 29.6 158A  35.4 17.5 85.2 159A  5.3 3.5 24.4 160A  33.2 13.9103.9 161A  7.4 3.7 11.7 162A  7.1 4.7 55.1 163A  4.8 4.8 28.5 164A 10.7 5.8 45.9 165A  21.9 8.0 113.9 166A  20.5 6.3 90.2 167A  23.7 7.761.0 168A  7.9 5.7 24.2 169A  34.5 7.6 144.4 170A  31.4 10.8 200.0 171A 6.1 6.1 27.7 172A  4.8 6.1 31.6 173A  104.3 37.9 174A  18.8 14.3 55.8175A  18.2 10.9 65.1 176A  6.4 4.9 29.0 177A  8.8 4.5 21.1 178A  4.9 7.547.7 179A  7.2 7.6 25.6 180A  9.3 4.9 16.8 181A  8.4 7.4 26.4 182A  8.07.1 22.3 183A  18.4 4.9 42.1 184A  28.9 5.3 94.8

Compounds of formula B showed the following activity in this assay: IC₅₀(MX-1), IC₅₀ (DLD-1), IC₅₀ (LNCap), Compound μM μM μM  1B 4.0 2.5 5.1 2B 200 0.3 10.9  3B 7.4 4.3 14.8  4B 4.1 2.9 11.5  6B 6.9 0.8 11.6  7B1.9 1.5 9.5  8B 10.9 6.5 28.0  9B 6.0 6.1 25.5 10B 10.8 6.1 1.9 11B 3.02.1 4.5 12B 4.3 1.9 7.1 13B 1.9 2.3 4.7 14B 2.3 3.2 5.7 15B 1.4 2.4 4.416B 2.9 4.5 9.2 17B 6.2 4.6 18.6 18B 8.4 2.7 49.8 19B 7.6 3.8 52.0 20B5.1 4.0 33.3 21B 14.9 4.1 131.9 22B 7.0 2.5 40.8 23B 1.1 0.7 3.8

The compound of formula 1C showed the following activity in this assay:IC₅₀ (MX-1), IC₅₀ (DLD-1), IC₅₀ (LNCap), Compound μM μM μM 1C 0.3 0.31.4

In vitro Example 2 Cell Cycle Analysis

Log-phase DLD-1 cells were seeded in a 75-mL flask for several hours toallow cell attachment, with the seeding density chosen so that the cellculture would be less than 80% confluent on the day of harvest. The testcompounds were added (dissolved in DMSO) at approximately IC₇₅₋₈₅ toachieve a final DMSO concentration of 0.1%, and the cells then incubatedfurther for one and two days. Following incubation, the cells wereharvested by trypsinization, fixed in 75% aqueous ethanol, and stored at−20° C. until further analysis. To determine the cellular DNA content,which reflects the cell cycle status, the fixed cells were washed twicewith phosphate-buffered saline and then treated with RNase for 30minutes at 37° C. They were then stained with the fluorescent dyepropidium iodide, followed by FACS analysis on a Becton DickinsonFACSCalibur system. Similarly to canfosfamide, and in contrast tomelphalan, both compounds 70A and 128A induced a G2/M cycle block inthis assay. Other compounds of formula A and formula B were tested inthis assay.

In vitro Example 3 DNA Damage Assay

Lambda DNA (1 μg in 20 μL phosphate-buffered saline) was incubated withthe test compounds (concentrations of 100 μM and 300 μM, dissolved inDMSO) at room temperature for one day, and the DNA then purified withthe DNeasy kit. After quantitation by a plate assay using the SYBR-Goldassay (5 μL DNA+200 μL dye at 1:10,000 dilution in TE buffer), 250 pg ofeach sample were amplified by PCR using AmpliTaq Gold reagents and theprimers 5′-ccg act ggc acc gct tt-3′ and 5′-cag gcc acc atc acg cat-3′,with PCR parameters 95° C., 10 min; 25 cycles of (95° C., 30 sec; 72°C., 3 min); 72° C., 10 min; then holding the samples at 4° C. untilanalysis. The PCR products were quantitated by the same SYBR-Gold assay.Similarly to canfosfamide, and in contrast to melphalan, both compounds70A and 128A showed only marginal DNA damage activity in this assay.Other compounds of formula A and formula B were tested in this assay,and many showed only low amounts of DNA damage when compared to agentssuch as melphalan.

In vitro Example 4 Cross-Resistance Assay

Cross-resistance between test compounds and anticancer agents areassayed by the use of matched cell line pairs, i.e. a standard cancercell line and a derivative cell line that has been made resistant to aparticular anticancer agent by culture in the presence of a sublethalconcentration of the agent. Standard cytotoxicity assays are performedin each cell line, essentially as described in Example 16, and theresistance ratio, the ratio of the IC₅₀ for the derivative cell line tothe IC₅₀ for the standard cell line is calculated. A test compound isconsidered cross-resistant with a selected anticancer agent if theresistance ratio for the test compound is approximately 2 or higher incells resistant to the selected anticancer agent. Compounds 70A and 128Awere not cross-resistant with doxorubicin in the P388 murine leukemiacell line. Other compounds of formula A and formula B were tested inthis assay, and many were also not cross-resistant.

In vitro Example 5 Synergism Assay

Synergism between test compounds and anticancer agents ate assayed in achosen cell line. Standard cytotoxicity assays are performed in the cellline, essentially as described in Example 16, using fixed or variableratios of the test compound and the anticancer agent, with enhancementof cytotoxicity when the test compound is combined with an anticanceragent compared to either compound alone indicating synergism. Theresults can also be analyzed using the Combination Index (CI) methodwith the “CalcuSyn” program from Biosoft, with a CI value less than 1indicating synergy, 1 indicating an additive effect, and greater than 1indicating antagonism. Similarly to canfosfamide, compound 128A was seento be synergistic with carboplatin in the OVCAR-3 cell line in thisassay.

In vivo Examples In vivo Example 1 MX-1 Xenograft Assay, IntraperitonealAdministration

Female athymic nu/nu mice (Harlan, Indianapolis, Ind., U.S.A. or similarvendor), 6-8 weeks old (approximately 20 g), were implanted in themammary fat pad of the right fore flank with 20-30 mg pieces of MX-1(human breast cancer) tumor harvested from similar mice that hadpreviously been implanted with the MX-1 tumor. Approximately 7-10 daysafter tumor transplantation, when the tumor weight was approximately50-200 mg, the mice were assigned to treatment groups. Groups of micewere treated with compounds 5A, 13A, 15A, 23A, 70A, 128A, 171A, 172A,and 180A at 100 mg/Kg by intraperitoneal injection once/day for 5, 9, or14 days, with vehicle control. All compounds were active in this assay,with tumor growth inhibition compared to vehicle between 5% (compound171A) and 99% (compound 13A), with most compounds causing at least 30%tumor growth inhibition. Compounds 13A, 70A, and 128A were also testedover a range of doses and treatment times, and caused dose-dependenttumor inhibition. Compound 13A was tested at 25, 50, or 100 mg/Kg for 5or 9 days, causing tumor inhibition between 19% (25 mg/Kg for 5 days)and 99% (100 mg/Kg for 9 days). Compound 70A was tested at 50, 100, and150 mg/Kg for 5 or 12 days, causing tumor inhibition between 16% (50mg/Kg for 5 days) and 95% (150 mg/Kg for 12 days).

In vivo Example 2 MX-1 Xenograft Assay, Oral Administration

A study similar to that described in In vivo Example 1 was performedusing oral administration of compounds 13A and 70A. Groups of mice weretreated with compound 13A at 50, 100, or 300 mg/Kg, or compound 70A at100, 150, 200, or 250 mg/Kg, by gavage once/day for 5 days (with vehiclecontrol). Tumor growth inhibition was measured 13 days after the startof treatment. Both compounds were active in this assay and causeddose-dependent tumor inhibition. Compound 13A caused a dose-dependentinhibition of tumor growth between 63% (50 mg/Kg) and 100% (300 mg/Kg)compared to vehicle, and compound 70A caused a dose-dependent inhibitionbetween 36% (at 100 mg/Kg) and 96% (250 mg/Kg).

In vivo Example 3 HCT116 Xenograft Assay, Intraperitoneal Administration

Male athymic nu/nu mice, 6-8 weeks old (approximately 20 g), wereimplanted subcutaneously in the right fore flank with 20-30 mg pieces ofHCT116 (human colon cancer) tumor harvested from similar mice that hadpreviously been implanted with the HCT116 tumor. Approximately 7-10 daysafter tumor transplantation, when the tumor weight was approximately50-200 mg, the mice were assigned to treatment groups. Groups of micewere treated with compounds 5A or 13A at 100 mg/Kg, or compound 70A at150 mg/Kg, by intraperitoneal injection once/day for 11 days, withvehicle control. Tumor growth inhibition was measured 13 days after thestart of treatment. All three compounds were active in this assay, withcompound 5A causing 10% inhibition of tumor growth compared to vehicle,compound 13A causing 43% inhibition, and compound 70A causing 26%inhibition. A similar study of compounds 13A and 70A, using tumorsstarted from tissue culture with 14 day compound administration andmeasurement 21 days after start of treatment, gave similar results.

In vivo Example 4 MiaPaCa-2 Xenograft Assay, IntraperitonealAdministration

Male athymic nu/nu mice, 6-8 weeks old (approximately 20 g), wereimplanted subcutaneously in the right fore flank with 20-30 mg pieces ofMiaPaCa-2 (human pancreatic cancer) tumor harvested from similar micethat had previously been implanted with the MiaPaCa-2 tumor.Approximately 7-10 days after tumor transplantation, when the tumorweight was approximately 50-200 mg, the mice were assigned to treatmentgroups. Groups of mice were treated with compound 13A or 70A at 100mg/Kg by intraperitoneal injection once/day for 14 days, with vehiclecontrol. Tumor growth inhibition was measured 15 days after the start oftreatment. Both compounds were active in this assay, with compound 13Acausing 78% inhibition of tumor growth compared to vehicle and compound70A causing 38% inhibition.

In vivo Example 5 MiaPaCa-2 Xenograft Assay, Oral Administration

A study similar to that described in In vivo Example 4, but using oraladministration of compounds 13A and 70A at 200 mg/Kg for 7 days, gavesimilar results to in vivo Example 4. Both compounds were active in thisassay, with compound 13A causing 82% inhibition of tumor growth andcompound 70A causing 27% inhibition.

All compounds tested were safe at the doses tested.

Formulation and Therapeutic Examples Formulation Example 1 Formulationfor Oral Administration

A solid formulation for oral administration is prepared by combining thefollowing: Compound of this invention 25.0% w/w Magnesium stearate 0.5%w/w Starch 2.0% w/w Hydroxypropylmethylcellulose 1.0% w/wMicrocrystalline cellulose 71.5% w/wand the mixture is compressed to form tablets or filled into hardgelatin capsules containing, for example, 100 mg of the compound of thisinvention. Tablets may be coated, if desired, by applying a suspensionof a film-forming agent (for example, hydroxypropylmethylcellulose),pigment (for example, titanium dioxide), and plasticizer (for example,diethyl phthalate), and drying the film by evaporation of the solvent.

Formulation Example 2 Formulation for IV Administration

A formulation for IV administration is prepared by dissolving a compoundof this invention, for example as a pharmaceutically acceptable salt, toa concentration of 1% w/v in phosphate-buffered saline; and the solutionis sterilized, for example by sterile filtration, and sealed in sterilecontainers containing, for example, 100 mg of a compound of thisinvention.

Alternatively, a lyophilized formulation is prepared by dissolving acompound of this invention, again for example as a pharmaceuticallyacceptable salt, in a suitable buffer, for example the phosphate bufferof the phosphate-buffered saline mentioned above, sterilizing thesolution and dispensing it into suitable sterile vials, lyophilizing thesolution to remove the water, and sealing the vials. The lyophilizedformulation is reconstituted by the addition of sterile water, and thereconstituted solution may be further diluted for administration with asolution such as 0.9% sodium chloride intravenous infusion or 5%dextrose intravenous infusion.

Therapeutic Example Therapy with compounds of this Invention

A compound of this invention, diluted in 5% dextrose intravenousinfusion, is administered intravenously over 30 minutes to a patientsuffering from metastatic ovarian carcinoma at an initial dose of 100mg/m²; and this dose is increased to 250 mg/M², 500 mg/m², 750 mg/m²,and 1000 mg/m². The compound is administered at 1-week intervals. Thesame dose escalation is administered at 2- and 3-week intervals to otherpatients suffering from the same cancer.

While this invention has been described in conjunction with specificembodiments and examples, it will be apparent to a person of ordinaryskill in the art, having regard to that skill and this disclosure, thatequivalents of the specifically disclosed materials and methods willalso be applicable to this invention; and such equivalents are intendedto be included within the following claims.

1. A compound of formula A, formula B, or formula C:

where: each R is independently hydrogen, C₁₋₆ alkyl, or —CH₂CH₂X, whereeach X is independently Cl, Br, C₁₋₆ alkanesulfonyloxy, halo-C₁₋₆alkanesulfonyloxy, or benzenesulfonyloxy optionally substituted with upto three substituents selected from halo, C₁₋₃ alkyl, halo-C₁₋₃ alkyl,C₁₃ alkyloxy, or halo-C₁₋₃ alkyloxy, provided that at least two R's ineach phosphorodiamidate group are —CH₂CH₂X; R¹ is optionally substitutedalkyl, optionally substituted heteroalkyl, optionally substituted aryl,optionally substituted aralkyl, optionally substituted heteroaryl, oroptionally substituted heteroaralkyl; and R² is optionally substitutedalkanediyl, optionally substituted heteroalkanediyl, optionallysubstituted arenediyl, optionally substituted arenedialkyl, optionallysubstituted heteroarenediyl, or optionally substitutedheteroarenedialkyl, or its salt.
 2. The compound of claim 1 where each Ris —CH₂CH₂X.
 3. The compound of claim 2 where each X is Cl, Br,methanesulfonyloxy, trifluoromethanesulfonyloxy, benzenesulfonyloxy, or4-toluenesulfonyloxy.
 4. The compound of claim 3 where each X is Cl. 5.The compound of claim 1 that is a compound of formula A or its salt. 6.The compound of claim 5 where R¹ is substituted with one or more groupsthat enhance the solubility of the compound over a compound that is notso substituted.
 7. The compound of claim 5 where R¹ is optionallysubstituted alkyl.
 8. The compound of claim 5 where R¹ is the residue ofan optionally protected thiol-amino acid or of an elaborated thiol-aminoacid.
 9. The compound of claim 5 where R¹ is an optionally elaborated2-mercaptoacetic acid or 3-mercaptopropionic acid.
 10. The compound ofclaim 5 where R¹ is optionally substituted aralkyl.
 11. The compound ofclaim 10 where R¹ is optionally substituted benzyl.
 12. The compound ofclaim 1 that is selected from:[2-[[bis[bis(2-chloroethyl)amino]phosphinyl]oxy]ethyl]sulfonylbutane,N-methyl-2-[[2-[[bis[bis(2-chloroethyl)amino]phosphinyl]oxy]ethyl]sulfonyl]acetamide,N-{2-[[2-[[bis[bis(2-chloroethyl)amino]phosphinyl]oxy]ethyl]sulfonyl]acetyl}-L-phenylalanineN-[2-(4-morpholino)ethyl]-2-[[2-[[bis[bis(2-chloroethyl)amino]phosphinyl]oxy]ethyl]sulfonyl]acetamide,3-[[2-[[bis[bis(2-chloroethyl)amino]phosphinyl]oxy]ethyl]sulfonyl]-L-alanine,α-[[2-[[bis[bis(2-chloroethyl)amino]phosphinyl]oxy]ethyl]sulfonyl]toluene,α-[[2-[[bis[bis(2-chloroethyl)amino]phosphinyl]oxy]ethyl]sulfonyl]-4-{[4-(2-thenoyl)-1-piperazinyl]carbonyl}toluene,α-[[2-[[bis[bis(2-chloroethyl)amino]phosphinyl]oxy]ethyl]sulfonyl]-4-[(ethoxycarbonylmethyl)-aminocarbonyl]toluene,and2-[[[2-[[bis[bis(2-chloroethyl)amino]phosphinyl]oxy]ethyl]sulfonyl]methyl]thiophene,and their salts.
 13. The compound of claim 12 that isN-methyl-2-[[2-[[bis[bis(2-chloroethyl)amino]-phosphinyl]oxy]ethyl]sulfonyl]acetamide.14. The compound of claim 12 that is3-[[2-[[bis[bis(2-chloroethyl)amino]phosphinyl]oxy]-ethyl]sulfonyl]-L-alanineor its salt.
 15. The compound of claim 1 that is a compound of formula Bor its salt.
 16. The compound of claim 15 where R² is substituted withone or more groups that enhance the solubility of the compound over acompound that is not so substituted.
 17. The compound of claim 1 that isa compound of formula C or its salt.
 18. A pharmaceutical compositioncomprising a compound of claim 1 and an excipient.
 19. A pharmaceuticalcomposition comprising a compound of claim 12 and an excipient.
 20. Apharmaceutical composition comprising a compound of claim 13 and anexcipient.
 21. A pharmaceutical composition comprising a compound ofclaim 14 and an excipient.
 22. A method of treating cancer in a humancomprising administering to the human a compound of claim 1 or apharmaceutical composition of claim
 18. 23. A method of preparing acompound of formula A, formula B, or formula C:

where: each R is independently hydrogen, C₁₋₆ alkyl, or —CH₂CH₂X, whereeach X is independently Cl, Br, C₁₋₆ alkanesulfonyloxy, halo-C₁₋₆alkanesulfonyloxy, or benzenesulfonyloxy optionally substituted with upto three substituents selected from halo, C₁₋₃ alkyl, halo-C₁₋₃ alkyl,C₁₋₃ alkyloxy, or halo-C₁₋₃ alkyloxy, provided that at least two R's ineach phosphorodiamidate group are —CH₂CH₂X; R¹ is optionally substitutedalkyl, optionally substituted heteroalkyl, optionally substituted aryl,optionally substituted aralkyl, optionally substituted heteroaryl, oroptionally substituted heteroaralkyl; and R² is optionally substitutedalkanediyl, optionally substituted heteroalkanediyl, optionallysubstituted arenediyl, optionally substituted arenedialkyl, optionallysubstituted heteroarenediyl, or optionally substitutedheteroarenedialkyl, or its salt, comprising: (a) oxidation of thecorresponding compound of formula AA, formula BB, or formula CC:

with any reactive moiety in R¹ or R² protected against oxidation ifnecessary; optionally followed by one or more of: (b) deprotection of aprotected compound of formula A, formula B, or formula C; (c) conversionof the R groups of a compound of formula A, formula B, or formula C intoother R groups to form another compound of formula A, formula B, orformula C; (d) elaboration of the R¹ group of a compound of formula A orthe R² group of the compound of formula B by synthetic methods known perse to form another compound of formula A or formula B; (e) formation ofa salt of a compound of formula A, formula B, or formula C; (f)conversion of a salt of a compound of formula A, formula B, or formula Cto another salt of formula A, formula B, or formula C; and (g)conversion of a salt of a compound of formula A, formula B, or formula Cto a non-salt form of the compound of formula A, formula B, or formulaC.
 24. A compound of formula BB or formula CC:

where: each R is independently hydrogen, C₁₋₆ alkyl, or —CH₂CH₂X, whereeach X is independently Cl, Br, C₁₋₆ alkanesulfonyloxy, halo-C₁₋₆alkanesulfonyloxy, or benzenesulfonyloxy optionally substituted with upto three substituents selected from halo, C₁₋₃ alkyl, halo-C₁₋₃ alkylC₁₋₃ alkyloxy, or halo-C₁₋₃ alkyloxy, provided that at least two R's ineach phosphorodiamidate group are —CH₂CH₂X; and R² is optionallysubstituted alkanediyl, optionally substituted heteroalkanediyl,optionally substituted arenediyl, optionally substituted arenedialkyl,optionally substituted heteroarenediyl, or optionally substitutedheteroarenedialkyl, or its salt.
 25. The compound of claim 24 where eachR is —CH₂CH₂X.
 26. The compound of claim 25 where each X is Cl, Br,methanesulfonyloxy, trifluoromethanesulfonyloxy, benzenesulfonyloxy, or4-toluenesulfonyloxy.
 27. The compound of claim 26 where each X is Cl.28. The compound of claim 24 that is a compound of formula BB or itssalt.
 29. The compound of claim 24 that is a compound of formula CC orits salt.
 30. A pharmaceutical composition comprising a compound ofclaim 24 and an excipient.
 31. A method of treating cancer in a humancomprising administering to the human a compound of claim 24 or apharmaceutical composition of claim 30.